Optimisation of Mix Proportion of 3D Printable Mortar Based on Rheological Properties and Material Strength Using Factorial Design of Experiment.

In the production of 3D printable mortar (3DPM), numerous efforts have been made globally to effectively utilise various cementitious materials, admixtures, and fibres. The determination of rheological and material strength properties is crucial for successful 3D concrete printing because the materials used in 3DPM must possess the unique characteristic of making mortar flowable while being strong enough to support the weight of subsequent layers in both fresh and hardened states. The complexity of the required characteristics makes it challenging to develop an optimised mix composition that satisfies both the rheological and material strength requirements, given the wide range of available admixtures, supplementary cementitious materials, and fibres. Fly ash, basalt fibre and superplasticiser when blended with cement can help to improve the overall performance of 3DPM. The objective of this research is to optimise the rheological properties and material strength of 3D printable mortars (3DPM) containing cement, fly ash, basalt fibre, and superplasticiser. This study aims to produce 3DPM with an optimised mix composition to meet the requirements of both rheological and material strength characteristics using the factorial design approach and desirability function. Different dosages of cement, fly ash, basalt fibre, and superplasticiser are chosen as the primary design parameters to develop statistical models for the responses of rheological and material strength properties at 7 and 28 days. The results expressed in terms of the measured properties are valid for mortars made with cement ranging from 550 to 650 kg/m3, fly ash from 5% to 20% (of cement), superplasticiser from 2 to 4 kg/m3, and basalt fibre from 1 to 3 kg/m3. The rheological properties are evaluated using slump flow, cone penetrometer, and cylindrical slump tests, while the mechanical strength is evaluated using a three-point bending test and compressive test. A full factorial design experiment (FoE) is used to determine the significant parameters effecting the measured properties. Prediction models are developed to express the measured properties in terms of the primary parameters. The influence of cement, fly ash, basalt fibre, and superplasticiser is analysed using polynomial regression to determine the main effects and interactions of these primary parameters on the measured properties. The results show that the regression models established by the factorial design approach are effective and can accurately predict the performance of 3DPM. Cement, fly ash, and superplasticiser dosages have significant effects on the rheological and mechanical properties of mortar, while basalt fibre is able to influence the static yield stress and flexural strength of 3DPM. The utilisation of regression models and isoresponse curves allows for the identification of significant trends and provides valuable insight into the behaviour of the material, while desirability function is useful to optimise overall performance of mix proportions to meet the desired performance objective at fresh and hardened states.

Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity.

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg-1). Among samples containing more than 25 µg kg-1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg-1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues.

Gravimetric quantitative validation of botanic impurities in feed.

BACKGROUND: Harmful botanical impurities may contaminate feed and feed materials and be a potential danger to animal or human health, or to the environment. The aim of this study was to establish rapid and sensitive methods that can be used in routine official controls to determine botanical impurities such as Datura stramonium, Ricinus communis, Crotaliaria spp., and Ambrosia spp. in animal feed and raw materials. Claviceps sclerotia were also detected in cereals, due to the similarities of the targets and the analytical procedure. Regulation (EU) 625/2017, which replaces Reg. 2004/882/EC, states that EU member states should conduct official controls in assessed and accredited laboratories and that the analytical methods must be validated before use by considering parameters such as specificity, precision, recovery, and measurement uncertainly. RESULTS AND CONCLUSION: The results demonstrate that all of the methods tested are suitable for the official quantitative analyses required by EU official legislation. © 2020 Society of Chemical Industry.

Detection of insect's meal in compound feed by Near Infrared spectral imaging.

Insects have recently emerged as a new protein source for both food and feed. Some studies have already demonstrated that insects' meal can be successfully added to animal feed without threaten animals' growth indices. However, effective and validated tests to individuate insects' meal in feed are strongly needed to meet traceability and safety concerns and to support the European legislation under development. Spectroscopic techniques represent valuable rapid and non-destructive methods that can be applied for in-situ analysis in feed production plants or in farms. In this work a Fourier Transform Near Infrared spectroscopy imaging (FT NIR) as a potential screening method for the detection and quantification of insects' meal in feed is presented. Discriminant analysis was used for the automatic recognition of insects' meal fragments into the feed matrix. Moreover, the possibility to quantify insect's meal in feed sample was successfully tested. The proposed method is a rapid and green strategy for feed contamination screening analysis.

Gravimetric quantitative determination of packaging residues in feed from former food.

Valorisation of former foodstuff products (FFP) in feed is part of a long-term strategy for sustainability. An approach to valorise FFP outside the waste value chain is their use as an alternative source of feed materials, with a subsequent optimisation of the environmental impact of products. In the current practice of food production, food packaging is provided to ensure the maintenance of food quality and safety during transport and storage. One of the problems of reusing FFP is how to deal with packaging materials or remains that can become residues in the feed. The aim of this study is to propose a fast and sensitive gravimetric method, fit for routine official controls, for the determination of packaging residues in feed. The developed method can briefly be summarised as: (1) visual selection of the undesired ingredients which can be identified as remnants of packaging materials; (2) weighing of the selected materials; (3) defatting; (4) dehydration; (5) final weighing; and (6) reporting of weight and percentage. Moreover, the method has been validated through the determination of some of the parameters listed in Council Regulation 2004/882/EC (i.e., specificity, limit of quantification (LOQ), recovery, repeatability, within-laboratory reproducibility and measurement uncertainty).

Species-specific detection of processed animal proteins in feed by Raman spectroscopy.

The existing European Regulation (EC n° 51/2013) prohibits the use of animals meals in feedstuffs in order to prevent Bovine Spongiform Encephalopathy infection and diffusion, however the legislation is rapidly moving towards a partial lifting of the "feed ban" and the competent control organisms are urged to develop suitable analytical methods able to avoid food safety incidents related to animal origin products. The limitations of the official methods (i.e. light microscopy and Polymerase Chain Reaction) suggest exploring new analytic ways to get reliable results in a short time. The combination of spectroscopic techniques with optical microscopy allows the development of an individual particle method able to meet both selectivity and sensitivity requirements (0.1%w/w). A spectroscopic method based on Fourier Transform micro-Raman spectroscopy coupled with Discriminant Analysis is here presented. This approach could be very useful for in-situ applications, such as customs inspections, since it drastically reduces time and costs of analysis.

In vitro activity of MSI-78 alone and in combination with antibiotics against bacteria responsible for bloodstream infections in neutropenic patients.

MSI-78 is a 22 amino acid amphipathic peptide with potent antimicrobial activity against Gram-positive and Gram-negative organisms, including antibiotic-resistant strains. In this study, we assessed the in vitro activity of MSI-78 alone and in combination with eight clinically used antimicrobial agents against several strains of Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli isolated from blood of neutropenic febrile patients. Antimicrobial activity of MSI-78 was measured by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill studies and checkerboard titration method. The Gram-negative isolates were susceptible to the peptide at concentrations in the range 0.50-16 mg/L, while staphylococci showed lower susceptibility. MSI-78 demonstrated a higher antimicrobial activity than colistin against Gram-negative organisms. The checkerboard titration method demonstrated synergy when the peptide was combined with beta-lactams. These results provide evidence for the potential use of MSI-78 in the management of severe infections in neutropenic patients.

In vitro activity and killing effect of uperin 3.6 against gram-positive cocci isolated from immunocompromised patients.

The in vitro activity of uperin 3.6, alone or combined with six antibiotics, against gram-positive cocci, including Rhodococcus equi, methicillin-resistant staphylococci, and vancomycin-resistant enterococci, was investigated. All isolates were inhibited at concentrations of 1 to 16 mg/liter. Synergy was demonstrated when uperin 3.6 was combined with clarithromycin and doxycycline.

In vitro activity of citropin 1.1 alone and in combination with clinically used antimicrobial agents against Rhodococcus equi.

OBJECTIVES: The aim of this study was to investigate the in vitro activity of citropin 1.1, an antimicrobial peptide derived from the Australian tree frog Litoria citropa, alone and in combination with ampicillin, ceftriaxone, doxycycline, netilmicin, ciprofloxacin, rifampicin, linezolid, vancomycin, clarithromycin and imipenem against 12 nosocomial isolates of Rhodococcus equi. METHODS: Antimicrobial activity of citropin 1.1 was measured by MIC, MBC, time-kill studies and chequerboard titration method. RESULTS: All isolates were inhibited at concentrations of citropin 1.1 between 2 and 8 mg/L. Combination studies demonstrated synergy only when the peptide was combined with clarithromycin, doxycycline and rifampicin. CONCLUSIONS: Our findings show that citropin 1.1 is active against R. equi and that its activity could be enhanced when it is combined with hydrophobic antibiotics.

In vitro activity and killing effect of citropin 1.1 against gram-positive pathogens causing skin and soft tissue infections.

The in vitro activity of citropin 1.1 against gram-positive cocci was measured by MIC, minimal bactericidal concentration, time-kill studies, and a checkerboard titration method. Streptococci and staphylococci were inhibited at concentrations between 1 and 16 mg/liter, respectively. Enterococci showed less susceptibility. Synergy was demonstrated when citropin 1.1 was combined with clarithromycin and doxycycline.

In vitro activity of the histatin derivative P-113 against multidrug-resistant pathogens responsible for pneumonia in immunocompromised patients.

The in vitro activity of the histatin derivative P-113, alone or combined with eight antibiotics, was investigated against multidrug-resistant strains isolated from clinical specimens of immunocompromised patients with pneumonia. The gram-negative isolates were susceptible to P-113. S. aureus showed less susceptibility. Synergy was demonstrated when P-113 was combined with beta-lactams against gram-negative organisms.

In vitro activity and killing effect of temporin A on nosocomial isolates of Enterococcus faecalis and interactions with clinically used antibiotics.

OBJECTIVE: To study the in vitro activity of temporin A, a basic, highly hydrophobic, antimicrobial peptide amide derived from the skin of the European red frog Rana temporaria, alone and in combination with co-amoxiclav, imipenem, ciprofloxacin, linezolid and vancomycin, against 42 nosocomial isolates of Enterococcus faecalis. Fourteen of these were resistant to vancomycin. METHODS: Antimicrobial activity of temporin A was measured by MIC, MBC and time-kill studies and by the chequerboard titration method. RESULTS: All isolates were inhibited at concentrations of 1 to 16 mg/L. Combination studies carried out with E. faecalis ATCC 29212 and ATCC 51299 demonstrated synergy only when the peptide was combined with co-amoxiclav and imipenem. CONCLUSIONS: Our findings show that temporin A is active against E. faecalis and that its activity could be enhanced when it is combined with other antimicrobial agents.

RNAIII-inhibiting peptide improves efficacy of clinically used antibiotics in a murine model of staphylococcal sepsis.

RNAIII-inhibiting peptide (RIP, YSPWTNF-NH2) is a quorum-sensing peptide inhibitor that prevents Staphylococcus aureus toxin production and biofilm formation. A mouse sepsis model was used to test the efficacy of RIP alone or in combination with conventional antibiotics in suppressing S. aureus-induced sepsis. Mice were injected intravenously with 3.0x10(6)CFU of S. aureus ATCC 25923 or with 3.0x10(6)CFU of S. aureus strain Smith diffuse. All animals were randomized to receive intravenously isotonic sodium chloride solution as a control, or 20 mg/kg RIP alone or combined with 20 mg/kg cefazolin, 10 mg/kg imipenem, or 10 mg/kg vancomycin immediately or 6 h after bacterial challenge. Main outcome measures were bacteremia and lethality. All compounds reduced lethality when compared to controls. Although, in general combined-treated groups had significant lower bacterial counts when associated to singly-treated groups only the combination between RIP and vancomycin with respect to cefazolin gave a statistically significant decrease in the lethality rate. Lowest lethality rates (10%) and bacteremia (<10(2)CFU/ml) were obtained when RIP was administered in combination with vancomycin. Because RIP can be synergistic with current antibiotic therapies and help to reduce S. aureus exotoxins production, it can be considered a promising agent to associate with antibiotics for further clinical research into treatment of sepsis.

Effects of pexiganan alone and combined with betalactams in experimental endotoxic shock.

To investigate the efficacy of pexiganan, a 22-residue magainin analog, alone and combined with betalactmas antibiotics in three experimental rat models of Gram-negative septic shock. Adult male Wistar rats were given (i) an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS; (ii) 2x10(10)CFU of E. coli ATCC 25922; and (iii) intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg pexiganan, 1 mg/kg polymyxin B, 20 mg/kg imipenem, 60 mg/kg piperacillin alone and combined with 1 mg/kg pexiganan. Each group included 15 animals. Lethality, bacterial growth in blood or intra-abdominal fluid, endotoxin and TNF-alpha concentrations in plasma. All compounds reduced the lethality when compared to controls. Piperacillin and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. Pexiganan showed a slightly lower antimicrobial activity than betalactams even though it achieved a substantial higher decrease in endotoxin and TNF-alpha plasma concentrations than imipenem and piperacillin. No statistically significant differences were noted for antimicrobial and antiendotoxin activities between pexiganan and polymyxin B. Combination between pexiganan and betalactams showed to be the most effective treatment in reducing all variables measured. The use of a novel antimicrobial compound able to bind to LPS associated to potent antibiotics such as betalactams may become an important future consideration for sepsis treatment.

The antimicrobial peptide BMAP-28 reduces lethality in mouse models of staphylococcal sepsis.

OBJECTIVE: A mouse model of staphylococcal sepsis was used to compare the efficacy of the bovine antimicrobial peptide BMAP-28, a compound of the cathelicidin family, with that of conventional antibiotics. DESIGN: Prospective, randomized, controlled animal study. SETTING: Research laboratory in a university hospital. SUBJECTS: BALB/c male mice. INTERVENTIONS: BALB/c mice were injected intravenously with 2.0 x 10(6) colony-forming units of live Staphylococcus aureus ATCC 25923 or with 5.0 x 10(8) heat-killed cells of the same strain. All animals were randomized to receive intravenously isotonic sodium chloride solution, 2 mg/kg BMAP-28, 7 mg/kg imipenem, 7 mg/kg vancomycin, 7 mg/kg clindamycin, and 7 mg/kg clarithromycin immediately and at 6 hrs after bacterial challenge. MEASUREMENTS AND MAIN RESULTS: Lethality, quantitative blood cultures, and detection of tumor necrosis factor-alpha and interleukin-6 plasma levels. In the experiments performed with live bacteria, all compounds reduced lethality rates and bacterial growth compared with controls. Imipenem and vancomycin exhibited the highest efficacy on these main outcome measures. In the experiments performed using heat-killed organisms, only BMAP-28 demonstrated significant efficacy on lethality rates, tumor necrosis factor-alpha, and interleukin-6 plasma levels compared with controls. CONCLUSION: These results highlight the capacity of BMAP-28 to reduce the effects of components of the bacterial cells and suggest that it may be beneficial in the treatment of severe staphylococcal infections in concert with other antimicrobial agents.

Therapeutic efficacy of the magainin analogue MSI-78 in different intra-abdominal sepsis rat models.

OBJECTIVES: This study was designed to investigate the antimicrobial and anti-endotoxin activity of MSI-78, a synthetic cationic peptide analogue of magainin 2. METHODS: The in vitro antimicrobial activity of MSI-78 was investigated against the commercially available quality control strain Escherichia coli ATCC 25922. In addition, three rat models of septic shock were investigated: (i) rats were injected intraperitoneally with 1 mg Escherichia coli 0111:B4 LPS; (ii) rats were given an intraperitoneal injection of 2 x 10(10) cfu of Escherichia coli ATCC 25922; (iii) intra-abdominal sepsis was induced via caecal ligation and puncture. All animals were randomized to receive after 360 min intravenously isotonic sodium chloride solution, 1 mg/kg MSI-78, or 60 mg/kg piperacillin. Main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumour necrosis factor alpha (TNF-alpha) concentrations, and lethality. RESULTS: Our in vitro data showed that MSI-78 possesses a strong activity against Escherichia coli. The in vivo studies showed that all compounds reduced the lethality when compared to controls. MSI-78 showed a slightly higher antimicrobial activity than piperacillin and achieved a substantial decrease in endotoxin and TNF-alpha plasma concentrations than the beta-lactam. CONCLUSIONS: Because of its strong double anti-endotoxin and antimicrobial activities MSI-78 could be an interesting compound for Gram-negative septic shock treatment.

In vitro activity and killing effect of the synthetic hybrid cecropin A-melittin peptide CA(1-7)M(2-9)NH(2) on methicillin-resistant nosocomial isolates of Staphylococcus aureus and interactions with clinically used antibiotics.

The in vitro activity of CA(1-7)M(2-9)NH(2), a 15-residue synthetic hybrid peptide derived from the sequences of cecropin A and melittin, alone and in combination with amoxicillin-clavulanate, imipenem, clarithromycin, ciprofloxacin, rifampin, and vancomycin, was investigated against 40 nosocomial isolates of methicillin-resistant Staphylococcus aureus. Antimicrobial activity of CA(1-7)M(2-9)NH(2) was measured by minimal inhibitory concentration, MBC, and time-kill studies. All isolates were inhibited at concentrations of 1 to 16 microg/mL. Combination studies performed with S. aureusATCC 43300 demonstrated synergy only when CA(1-7)M(2-9)NH(2) was combined with amoxicillin-clavulanate and imipenem. Our findings show that CA(1-7)M(2-9)NH(2) is active against methicillin-resistant S. aureusand that its activity is enhanced when it is combined with several antimicrobial agents.

Temporin A soaking in combination with intraperitoneal linezolid prevents vascular graft infection in a subcutaneous rat pouch model of infection with Staphylococcus epidermidis with intermediate resistance to glycopeptides.

The efficacy of linezolid and temporin A in the prevention of prosthetic graft infection due to methicillin-resistant Staphylococcus epidermidis with intermediate resistance to glycopeptides was investigated in a subcutaneous rat pouch model. Linezolid and temporin A, alone or combined, greatly reduced the bacterial numbers compared to the effect with control drugs.

Potential therapeutic role of histatin derivative P-113d in experimental rat models of Pseudomonas aeruginosa sepsis.

BACKGROUND: Morbidity and mortality from Pseudomonas aeruginosa sepsis remain high despite the availability of antibiotics to which the microorganism is sensitive. METHODS: The in vitro activity of histatin derivative P-113d was investigated against Pseudomonas aeruginosa. In addition, its in vivo efficacy was studied in 3 rat models of infection: intraperitoneal injection of 1 mg of P. aeruginosa 10 lipopolysachharide, intraperitoneal injection of 2 x 10(10) cfu of P. aeruginosa ATCC 27853, and intra-abdominal sepsis induced by cecal ligation and puncture. Rats received isotonic sodium chloride solution parenterally (control groups), 1 mg of P-113d/kg of body weight, 1 mg of polymyxin B/kg of body weight, or 20 mg of imipenem/kg of body weight. Main outcomes measured were abdominal exudate and plasma bacterial growth, plasma concentrations of endotoxin and tumor necrosis factor (TNF)- alpha, and lethality. RESULTS: The in vivo studies showed that all compounds reduced lethality, when compared with results for the control group. Overall, P-113d exhibited a slightly lower antimicrobial activity than did imipenem, even though P-113d achieved a substantial decrease in plasma concentrations of endotoxin and TNF- alpha, compared with the imipenem. No statistically significant differences for antimicrobial and antiendotoxin activities were noted between P-113d and polymyxin B. DISCUSSION: These results provide evidence for double antiendotoxin and antimicrobial activity for P-113d and point to its potential use for the treatment of severe infections.

A chimeric peptide composed of a dermaseptin derivative and an RNA III-inhibiting peptide prevents graft-associated infections by antibiotic-resistant staphylococci.

Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices. Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms. RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms. K(4)-S4(1-13)(a) is a 13-residue dermaseptin derivative (DD(13)) believed to kill bacteria via membrane disruption. We tested each of these peptides as well as a hybrid construct, DD(13)-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis (MRSE). In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD(13)-RIP and DD(13) were equally effective, and that the chimeric peptide but not DD(13) was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis. In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose. In addition, each of the peptides acted synergistically with antibiotics. The data indicate that RIP and DD(13) act in synergy by attacking bacteria simultaneously by two different mechanisms. Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections.