The need for guidance in antidepressant drug development: Revisiting the role of the forced swim test and tail suspension test.

Depressive disorders are one of the most common mental disorders globally and progress in treating these disorders has been hampered, in part, by a lack of suitable nonclinical efficacy tests. Two common tests used in nonclinical efficacy studies of antidepressants-the forced swim test (FST) and tail suspension test (TST)-have come under criticism in recent years for their inconsistency and lack of validity, yet they continue to be used in the pharmaceutical industry. In this review, we provide a rationale for why international pharmaceutical regulatory and guidance agencies should begin issuing direction on methods for non-clinical efficacy testing that traditionally use the FST and TST, particularly considering that some regulators, such as those in the U.S. and E.U., allow the authorization of clinical trials to proceed without requiring tests in animals. The area of antidepressant drug discovery represents an important opportunity for reducing the attrition of psychiatric drugs, harmonizing regulatory requirements, and reducing animal use. Specific recommendations for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) have been provided.

Efficacy of Three Antimicrobials Against two SARS-COV-2 Surrogates, Bovine Coronavirus and Human Coronavirus OC43, on Hard or Soft Nonporous Materials.

The efficacy of three antimicrobials was evaluated against two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogates - bovine coronavirus (BCoV) and human coronavirus (HCoV) OC43 - on hard and soft nonporous materials. Three antimicrobials with three different active ingredients (chlorine, hydrogen peroxide, and quaternary ammonium compound + alcohol) were studied. Initially, a neutralization method was optimized for each antimicrobial. Then, we determined their efficacy against BCoV and HCoV OC43 in both suspension and on surfaces made with polyethylene terephthalate (PET) plastic and vinyl upholstery fabric. All tests were conducted under ambient environmental conditions with a soil load of 5% fetal bovine serum. After a 2-min exposure, all three antimicrobials achieved a >3.0 log10 reduction in viral titers in suspension. All three also reduced virus infectivity on both surface materials below the detection limit (0.6 log10 TCID50/carrier). Treatments in which the reduction in virus titer was <3.0 log10 were attributed to a decreased dynamic range on the carrier during drying prior to disinfection. The carrier data revealed that both surrogates were inactivated more rapidly (p <0.05) on vinyl or under conditions of high relative humidity. Three classes of antimicrobials were efficacious against both SARS-CoV-2 surrogate viruses, with BCoV demonstrating slightly less sensitivity compared to HCoV OC43. These findings also illustrate the importance of (1) optimizing the neutralization method and (2) considering relative humidity as a key factor for efficacy testing.

Antibacterial activity of solid surfaces is critically dependent on relative humidity, inoculum volume, and organic soiling.

Antimicrobial surface materials potentially prevent pathogen transfer from contaminated surfaces. Efficacy of such surfaces is assessed by standard methods using wet exposure conditions known to overestimate antimicrobial activity compared to dry exposure. Some dry test formats have been proposed but semi-dry exposure scenarios e.g. oral spray or water droplets exposed to ambient environment, are less studied. We aimed to determine the impact of environmental test conditions on antibacterial activity against the model species Escherichia coli and Staphylococcus aureus. Surfaces based on copper, silver, and quaternary ammonium with known or claimed antimicrobial properties were tested in conditions mimicking microdroplet spray or larger water droplets exposed to variable relative air humidity in the presence or absence of organic soiling. All the environmental parameters critically affected antibacterial activity of the tested surfaces from no effect in high-organic dry conditions to higher effect in low-organic humid conditions but not reaching the effect size demonstrated in the ISO 22169 wet format. Copper was the most efficient antibacterial surface followed by silver and quaternary ammonium based coating. Antimicrobial testing of surfaces using small droplet contamination in application-relevant conditions could therefore be considered as one of the worst-case exposure scenarios relevant to dry use surfaces.

Wound healing efficacy of a polymeric spray film solution containing Centella asiatica leaf extract on acute wounds.

OBJECTIVE: To study the clinical efficacy of a polymeric spray film containing Centella asiatica extract to heal acute wounds. METHOD: A polymeric spray film solution for wound healing was formulated using Centella asiatica extract, which contained triterpenes, including asiatic acid, madecassic acid, asiaticoside and madecassoside. The stability and physicochemical properties of the formulation were evaluated, and a multicentre, randomised, controlled trial was conducted to assess its clinical wound-healing efficacy. The Pressure Ulcer Scale for Healing (PUSH Tool) score was used to evaluate wound healing on days 0, 3, 5 and 7. RESULTS: The cohort consisted of 60 volunteers with clean-contaminated wounds (class 1), randomly assigned to the Control (n=30) and Testing (n=30) groups. The spray product contained asiatic acid, madecassic acid, asiaticoside and madecassoside at 0.20±0.02mg/ml, 0.16±0.01mg/ml, 0.32±0.03mg/ml and 0.10±0.00mg/ml, respectively. The pH value was 5.5±0.01, and the viscosity was 33±4cP. The product was stable for six months when stored at 30±2°C and at 40±2°C, in 75±5% relative humidity. The tested product significantly reduced the total PUSH and exudate scores, indicating that the polymeric spray film solution containing Centella asiatica improved wound healing. The average healing recovery times for the Testing and Control groups were 4.6±1.1 days and 4.87±1.0 days, respectively. CONCLUSION: In this study, Centella asiatica extract-containing polymeric spray film solution was beneficial as an acute wound medication, which could shorten healing time with no adverse effects.

Trypanosoma cruzi STIB980: A TcI Strain for Drug Discovery and Reverse Genetics.

Since the first published genome sequence of Trypanosoma cruzi in 2005, there have been tremendous technological advances in genomics, reverse genetics, and assay development for this elusive pathogen. However, there is still an unmet need for new and better drugs to treat Chagas disease. Here, we introduce a T. cruzi assay strain that is useful for drug research and basic studies of host-pathogen interactions. T. cruzi STIB980 is a strain of discrete typing unit TcI that grows well in culture as axenic epimastigotes or intracellular amastigotes. We evaluated the optimal parameters for genetic transfection and constructed derivatives of T. cruzi STIB980 that express reporter genes for fluorescence- or bioluminescence-based drug efficacy testing, as well as a Cas9-expressing line for CRISPR/Cas9-mediated gene editing. The genome of T. cruzi STIB980 was sequenced by combining short-read Illumina with long-read Oxford Nanopore technologies. The latter served as the primary assembly and the former to correct mistakes. This resulted in a high-quality nuclear haplotype assembly of 28 Mb in 400 contigs, containing 10,043 open-reading frames with a median length of 1077 bp. We believe that T. cruzi STIB980 is a useful addition to the antichagasic toolbox and propose that it can serve as a DTU TcI reference strain for drug efficacy testing.

Application of Medical Image Navigation Technology in Minimally Invasive Puncture Robot.

Microneedle puncture is a standard minimally invasive treatment and surgical method, which is widely used in extracting blood, tissues, and their secretions for pathological examination, needle-puncture-directed drug therapy, local anaesthesia, microwave ablation needle therapy, radiotherapy, and other procedures. The use of robots for microneedle puncture has become a worldwide research hotspot, and medical imaging navigation technology plays an essential role in preoperative robotic puncture path planning, intraoperative assisted puncture, and surgical efficacy detection. This paper introduces medical imaging technology and minimally invasive puncture robots, reviews the current status of research on the application of medical imaging navigation technology in minimally invasive puncture robots, and points out its future development trends and challenges.

Evaluation of temperature, drying time and other determinants for the recovery of Gram-negative bacterial pathogens in disinfectant efficacy testing.

BACKGROUND: In the clinical setting, surface disinfection is an important measure to reduce the risk of cross transmission of micro-organisms and the risk of nosocomial infections. Standardized methods can be used to evaluate disinfection procedures, as well as the effectiveness of the active ingredients used for disinfection. However, despite standardization, the results of such methodologies are still determined by several factors, and incorrect results may lead to invalid assumptions about the effectiveness of a disinfectant, posing significant health risks for patients and health personnel. AIM: The objective of this study was to evaluate several determinants for the recovery of Pseudomonas aeruginosa and other test organisms to establish their influence on the results of standardized disinfection methodologies, and to find Gram-negative strains that can be used as suitable replacements for P. aeruginosa. METHODS: The effects of inoculum application method, drying time, temperature and carrier material on the survival and recovery of the test organisms were evaluated using Student's t-test, one-way analysis of variance and Tukey's multiple comparison test. FINDINGS AND CONCLUSIONS: Temperature, drying time, application method and carrier material were found to affect the recovery of P. aeruginosa cells significantly, and therefore influence the outcome of the methodologies used. This study also showed thatP. aeruginosa could be replaced with the Gram-negative species Acinetobacter baumannii, a test organism used in many standardized methodologies, which responds better under the same circumstances and has a behaviour similar to that of P. aeruginosa in disinfectant efficacy tests.

Evaluation of Biofilm Cultivation Models for Efficacy Testing of Disinfectants against Salmonella Typhimurium Biofilms.

Within the European Union, Salmonella is frequently reported in food and feed products. A major route of transmission is upon contact with contaminated surfaces. In nature, bacteria such as Salmonella are often encountered in biofilms, where they are protected against antibiotics and disinfectants. Therefore, the removal and inactivation of biofilms is essential to ensure hygienic conditions. Currently, recommendations for disinfectant usage are based on results of efficacy testing against planktonic bacteria. There are no biofilm-specific standards for the efficacy testing of disinfectants against Salmonella. Here, we assessed three models for disinfectant efficacy testing on Salmonella Typhimurium biofilms. Achievable bacterial counts per biofilm, repeatability, and intra-laboratory reproducibility were analyzed. Biofilms of two Salmonella strains were grown on different surfaces and treated with glutaraldehyde or peracetic acid. Disinfectant efficacy was compared with results for planktonic Salmonella. All methods resulted in highly repeatable cell numbers per biofilm, with one assay showing variations of less than 1 log10 CFU in all experiments for both strains tested. Disinfectant concentrations required to inactivate biofilms were higher compared to planktonic cells. Differences were found between the biofilm methods regarding maximal achievable cell numbers, repeatability, and intra-laboratory reproducibility of results, which may be used to identify the most appropriate method in relation to application context. Developing a standardized protocol for testing disinfectant efficacy on biofilms will help identify conditions that are effective against biofilms.

Characterization of acute lung injury in the bleomycin rat model.

The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin-induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin-induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50-60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF-ß1, IL-1ß, TNF-α, IL-6, CINC-1, TIMP-1, and WISP-1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF-ß/Smad signaling pathway and increased expression of Galectin-3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin-induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

Pamoic acid is an inhibitor of HMGB1·CXCL12 elicited chemotaxis and reduces inflammation in murine models of Pseudomonas aeruginosa pneumonia.

BACKGROUND: High-mobility group box 1 protein (HMGB1) is an ubiquitous nuclear protein that once released in the extracellular space acts as a Damage Associated Molecular Pattern and promotes inflammation. HMGB1 is significantly elevated during Pseudomonas aeruginosa infections and has a clinical relevance in respiratory diseases such as Cystic Fibrosis (CF). Salicylates are HMGB1 inhibitors. To address pharmacological inhibition of HMGB1 with small molecules, we explored the therapeutic potential of pamoic acid (PAM), a salicylate with limited ability to cross epithelial barriers. METHODS: PAM binding to HMGB1 and CXCL12 was tested by Nuclear Magnetic Resonance Spectroscopy using chemical shift perturbation methods, and inhibition of HMGB1·CXCL12-dependent chemotaxis was investigated by cell migration experiments. Aerosol delivery of PAM, with single or repeated administrations, was tested in murine models of acute and chronic P. aeruginosa pulmonary infection in C57Bl/6NCrlBR mice. PAM efficacy was evaluated by read-outs including weight loss, bacterial load and inflammatory response in lung and bronco-alveolar lavage fluid. RESULTS: Our data and three-dimensional models show that PAM is a direct ligand of both HMGB1 and CXCL12. We also showed that PAM is able to interfere with heterocomplex formation and the related chemotaxis in vitro. Importantly, PAM treatment by aerosol was effective in reducing acute and chronic airway murine inflammation and damage induced by P. aeruginosa. The results indicated that PAM reduces leukocyte recruitment in the airways, in particular neutrophils, suggesting an impaired in vivo chemotaxis. This was associated with decreased myeloperoxidase and neutrophil elastase levels. Modestly increased bacterial burdens were recorded with single administration of PAM in acute infection; however, repeated administration in chronic infection did not affect bacterial burdens, indicating that the interference of PAM with the immune system has a limited risk of pulmonary exacerbation. CONCLUSIONS: This work established the efficacy of treating inflammation in chronic respiratory diseases, including bacterial infections, by topical delivery in the lung of PAM, an inhibitor of HMGB1.

Scientific Rationale for Waiving Clinical Efficacy Testing of Biosimilars.

After 18 years and the administration of billions of doses, there is little doubt about biosimilars' safety and efficacy. Yet, only 14 molecules in the EU and 9 in the US are available as biosimilars, among the 200+ targets, due mainly to the high development cost attributed to clinical efficacy testing after extensive analytical assessment, nonclinical testing, and clinical pharmacology comparisons. So far, none of the hundreds of clinical efficacy testing has failed because it cannot fail due to its lack of sensitivity for multiple reasons, as argued in this paper. This analysis is unique since biosimilars are the first category of products that are put to comparative testing as if these were new biological drugs. Clinical efficacy testing used to overcome differences in the analytical, nonclinical, and clinical pharmacology comparisons can lead to the approval of unsafe products. Only recently the regulatory agencies have begun to talk about this risk and shown their willingness to waive these studies. However, a clear change in the regulatory guidelines is required to change the mindset of all biosimilar stakeholders to bring a pivotal change in the availability of affordable biosimilars.

Novel Antibacterial Activity of Febuxostat, an FDA-Approved Antigout Drug against Mycobacterium tuberculosis Infection.

Accumulating evidence suggests that drug repurposing has drawn attention as an anticipative strategy for controlling tuberculosis (TB), considering the dwindling drug discovery and development pipeline. In this study, we explored the antigout drug febuxostat and evaluated its antibacterial activity against Mycobacterium species. Based on MIC evaluation, we found that febuxostat treatment significantly inhibited mycobacterial growth, especially that of Mycobacterium tuberculosis (Mtb) and its phylogenetically close neighbors, M. bovis, M. kansasii, and M. shinjukuense, but these microorganisms were not affected by allopurinol and topiroxostat, which belong to a similar category of antigout drugs. Febuxostat concentration-dependently affected Mtb and durably mediated inhibitory functions (duration, 10 weeks maximum), as evidenced by resazurin microtiter assay, time-kill curve analysis, phenotypic susceptibility test, and the Bactec MGIT 960 system. Based on these results, we determined whether the drug shows antimycobacterial activity against Mtb inside murine bone marrow-derived macrophages (BMDMs). Notably, febuxostat markedly suppressed the intracellular growth of Mtb in a dose-dependent manner without affecting the viability of BMDMs. Moreover, orally administered febuxostat was efficacious in a murine model of TB with reduced bacterial loads in both the lung and spleen without the exacerbation of lung inflammation, which highlights the drug potency. Taken together, unexpectedly, our data demonstrated that febuxostat has the potential for treating TB.

Data-Driven Identification of Biomarkers for In Situ Monitoring of Drug Treatment in Bladder Cancer Organoids.

Three-dimensional (3D) organoid culture recapitulating patient-specific histopathological and molecular diversity offers great promise for precision medicine in cancer. In this study, we established label-free imaging procedures, including Raman microspectroscopy (RMS) and fluorescence lifetime imaging microscopy (FLIM), for in situ cellular analysis and metabolic monitoring of drug treatment efficacy. Primary tumor and urine specimens were utilized to generate bladder cancer organoids, which were further treated with various concentrations of pharmaceutical agents relevant for the treatment of bladder cancer (i.e., cisplatin, venetoclax). Direct cellular response upon drug treatment was monitored by RMS. Raman spectra of treated and untreated bladder cancer organoids were compared using multivariate data analysis to monitor the impact of drugs on subcellular structures such as nuclei and mitochondria based on shifts and intensity changes of specific molecular vibrations. The effects of different drugs on cell metabolism were assessed by the local autofluorophore environment of NADH and FAD, determined by multiexponential fitting of lifetime decays. Data-driven neural network and data validation analyses (k-means clustering) were performed to retrieve additional and non-biased biomarkers for the classification of drug-specific responsiveness. Together, FLIM and RMS allowed for non-invasive and molecular-sensitive monitoring of tumor-drug interactions, providing the potential to determine and optimize patient-specific treatment efficacy.

Confirmatory efficacy testing for individual dose-placebo comparisons using serial gatekeeping procedure in dose-finding trials with multiple comparison procedures-modeling.

Dose-finding trials play a key role in the entire drug development process to determine optimal doses for regulatory approval. We address confirmatory efficacy testing for individual dose-placebo comparisons in the context of a dose-finding trial designed with multiple comparison procedures-modeling (MCP-Mod). An extension of the MCP-Mod, called closed MCP-Mod, has been proposed to carry out the MCP-Mod in conjunction with pairwise dose-placebo comparisons; however, an issue associated with the misspecification of candidate dose-response models remains. We consider another way to combine the MCP-Mod and the individual dose-placebo comparisons using serial gatekeeping procedures with fixed sequence, Holm, Hochberg, and step-down Dunnett procedure. The method controls the family-wise error rate in the strong sense and is simple enough to be implemented by existing software. Simulation studies suggested that the serial gatekeeping procedure was comparable with the closed MCP-Mod in terms of statistical power to detect the efficacy of at least one dose, and both methods were capable of pursuing the efficacy claim rather than just establishing the dose-response signal with less than a 20% increase in sample size when assuming monotonic dose-response shapes. The serial gatekeeping procedure would have advantages in the simplicity of implementation and ease of interpretation. The dose-finding trials aiming to declare the dose-response signal, as well as the efficacy of individual doses, would be worth considering as an option to accelerate the drug development program in certain situations.

3D Printing: Advancement in Biogenerative Engineering to Combat Shortage of Organs and Bioapplicable Materials.

Abstract: Organ or cell transplantation is medically evaluated for end-stage failure saving or extending the lives of thousands of patients who are suffering from organ failure disorders. The unavailability of adequate organs for transplantation to meet the existing demand is a major challenge in the medical field. This led to day-day-increase in the number of patients on transplant waiting lists as well as in the number of patients dying while on the queue. Recently, technological advancements in the field of biogenerative engineering have the potential to regenerate tissues and, in some cases, create new tissues and organs. In this context, major advances and innovations are being made in the fields of tissue engineering and regenerative medicine which have a huge impact on the scientific community is three-dimensional bioprinting (3D bioprinting) of tissues and organs. Besides this, the decellularization of organs and using this as a scaffold for generating new organs through the recellularization process shows promising results. This review discussed about current approaches for tissue and organ engineering including methods of scaffold designing, recent advances in 3D bioprinting, organs regenerated successfully using 3D printing, and extended application of 3D bioprinting technique in the field of medicine. Besides this, information about commercially available 3D printers has also been included in this article. Lay Summary: Today's need for organs for the transplantation process in order to save a patient's life or to enhance the survival rate of diseased one is the prime concern among the scientific community. Recent, advances in the field of biogenerative engineering have the potential to regenerate tissues and create organs compatible with the patient's body. In this context, major advances and innovations are being made in the fields of tissue engineering and regenerative medicine which have a huge impact on the scientific community is three-dimensional bioprinting (3D bioprinting) of tissues and organs. Besides this, the decellularization of organs and using this as a scaffold for generating new organs through the recellularization process shows promising results. This review dealt with the current approaches for tissue and organ engineering including methods of scaffold designing, recent advances in 3D bioprinting, organs regenerated successfully using 3D printing, and extended application of 3D bioprinting technique in the field of medicine. Furthermore, information about commercially available 3D printers has also been included in this article.

Construction and evaluation of a bioluminescent Pseudomonas aeruginosa reporter for use in preservative efficacy testing.

Preservative efficacy testing (PET) is a fundamental practice in industrial microbiology used to ensure product shelf-life and quality. To improve on current growth-based PET, bioluminescence was evaluated as a real-time bacterial viability indicator using Pseudomonas aeruginosa. Random mutagenesis of an industrial P. aeruginosa strain with a promoter-less luxCDABE mini-Tn5 was used to select a stable reporter (LUX12H5) with an un-altered growth and preservative susceptibility phenotype. Bioluminescence and viability were measured with and without preservatives (isothiazolinones, phenoxyethanol, and dimethyl dimethylol hydantoin) and an antibiotic comparator (ciprofloxacin). In the absence of antimicrobials, a good correlation between bioluminescence and viability (r2=0.92) was established. However, metabolic inhibition by isothiazolinone preservatives caused a rapid decline in light output that did not correlate to a reduced viability. Conversely, after ciprofloxacin exposure, the decline in viability was greater than that of bioluminescence. A positive attribute of the bioluminescence was the early detection of metabolic recovery and re-growth of preservative injured bacteria. Overall, while initial bioluminescence read-outs were less suited to current PET requirements, it shows promise as an early, direct indicator of bacterial regrowth in the context of long-term evaluation of preservative efficacy.

The Efficacy of Ultrasonic Pest Repellent Devices against the Australian Paralysis Tick, Ixodes holocyclus (Acari: Ixodidae).

Ultrasonic pest repellers are often promoted as a means of protecting people and pets from the bites of hematophagous arthropods, such as ticks. However, to date, there has been no published research on the effectiveness of these devices against the Australian paralysis tick, Ixodes holocyclus Neumann. The purpose of this study was to test the effectiveness of nine ultrasonic devices against female I. holocyclus. Two arenas were constructed, one for the test (with the ultrasonic device) and one for the control (no device). Each arena had a test and an escape chamber, connected by a corridor. Twenty ticks were placed in each test chamber. After the ultrasonic device was operated for 1 h, the number of ticks in both chambers was recorded. Ten replicates were conducted for each device. The average number of ticks that moved from the test to the escape chamber was greater in all the test arenas, with three devices being statistically different from the control. However, the highest percent of ticks that escaped was only 19.5%. This amount is insufficient to offer adequate protection against tick bites and this study adds further weight to previous investigations that ultrasonic devices should not be employed in pest management.

Variables Affecting the Recovery of Acanthamoeba Trophozoites.

While the results of Acanthamoeba testing have been extensively published, laboratories conducting such testing are left to develop their own methods in the absence of a standardized methodology. The wide disparity of methods has resulted in equally inconsistent reported results for contact lens care (CLC) products. This study's objective was to determine the source of these discrepancies by evaluating basic Acanthamoeba biology and their impact on antimicrobial efficacy testing, including the ability of a recovery method to stimulate a single trophozoite to proliferate. Antimicrobial efficacy testing was conducted using well-published Acanthamoeba strains, storage conditions, and growth-based recovery methods. To identify variables that influence results, test solutions with low Acanthamoeba disinfection rates were utilized to prevent differences from being masked by high log reductions. In addition, single-cell proliferation assays were executed to understand the growth requirements to stimulate trophozoite propagation in two recovery methods. These studies indicated that both nutrient density (>106 CFU) and the length of plate incubation (at least 14 days) could significantly influence the accurate recovery of trophozoites. Together, this study emphasizes the need to understand how Acanthamoeba trophozoites biology can impact test methods to create divergent results.

A rapid model for developing dry surface biofilms of Staphylococcus aureus and Pseudomonas aeruginosa for in vitro disinfectant efficacy testing.

BACKGROUND: Bacterial biofilms persistent on dry environmental surfaces in healthcare facilities play an important role in the occurrence of healthcare associated infections (HAI). Compared to wet surface biofilms and planktonic bacteria, dry surface biofilms (DSB) are more tolerant to disinfection. However, there is no official method for developing DSB for in vitro disinfectant efficacy testing. The objectives of this study were to (i) develop an in vitro model of DSB of S. aureus and P. aeruginosa for disinfectant efficacy testing and (ii) investigate the effect of drying times and temperatures on DSB development. We hypothesized that a minimum six log10 density of DSB could be achieved on glass coupons by desiccating wet surface biofilms near room temperatures. We also hypothesized that a DSB produced by the model in this study will be encased in extracellular polymeric substances (EPS). METHODS: S. aureus ATCC-6538 and P. aeruginosa ATCC-15442 wet surface biofilms were grown on glass coupons following EPA MLB SOP MB-19. A DSB model was developed by drying coupons in an incubator and viable bacteria were recovered following a modified version of EPA MLB SOP MB-20. Scanning electron microscopy was used to confirm the EPS presence on DSB. RESULTS: Overall, a minimum of six mean log10 densities of DSB for disinfectant efficacy were recovered per coupon after drying at different temperatures and drying times. Regardless of strain, temperature and dry time, 86% of coupons with DSB were confirmed to have EPS. CONCLUSION: A rapid model for developing DSB with characteristic EPS was developed for disinfectant efficacy testing against DSB.

Evaluation of the Efficacy of Mycotoxin Modifiers and Mycotoxin Binders by Using an In Vitro Rumen Model as a First Screening Tool.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.