Preparation of Komagataeibacter xylinus Inoculum for Bacterial Cellulose Biosynthesis Using Magnetically Assisted External-Loop Airlift Bioreactor

The aim of this study was to demonstrate the applicability of a novel magnetically assisted external-loop airlift bioreactor (EL-ALB), equipped with rotating magnetic field (RMF) generators for the preparation of Komagataeibacter xylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200 × higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible water-related properties, mechanical strength, nano-fibrillar structure and total crystallinity index. The lack of any negative impact of inoculum preparation method using RMF-assisted EL-ALB on BC properties is of paramount value for its future applications, including use as a biomaterial in tissue engineering, wound healing, and drug delivery, where especially BC liquid capacity, nanostructure, crystallinity, and mechanical properties play essential roles.

In Vitro Simulation of Human Colonic Fermentation: A Practical Approach towards Models’ Design and Analytical Tools

The human colonic microbiota plays an important role in the food digestion process and has a key role in maintaining health status. This community of microbes is inter-individually different due to several factors that modulate its composition. Among them, diet is one of the most relevant, which, in turn, is affected by environmental, economic, and cultural considerations. These pieces of evidence have promoted the study of the influence of diet on gut microbiota and the development of in vitro models that simulate the colonic digestion of foods. This narrative review aims to present a technical approach of the in vitro gut models available to evaluate the impact of diet on human colonic microbiota. A description and comments on the main characteristics, parameters, applicability, faecal inoculum preparation, and analytical tools are made. Despite the progress of in vitro colonic digestion models and metaomic applicability in this research field, there are still some challenges to face due to the lack of a consensus on the methodologies to conduct in vitro colonic digestions and the need to integrate the metaomic data to fully understand the influence of food in human colonic microbiota.

Antimicrobial Activity of Azadirachta indica, against Target Pathogens and Its Utility as a Disinfectant and Floor Cleaner

BACKGROUND Azadirachta indica, commonly known as neem, neem tree or Indian lilac, Limbo, Nim, Nimba, Medusa and Vempu. It is typically grown in tropical and subtropical regions. Neem belongs to family meliaceae. Neem is a native tree of India. It is also called “village pharmacy of south Asia” (India) because of its enormous medicinal properties and people use it most of the time without knowing its multiple advantages. This study was done to know the efficacy of medicinal plant named Azadirachta indica (Neem) against pathogenic microorganisms and about its utility as disinfectant and floor cleaner. METHODS Agar well diffusion method, Culture plate method, inoculum preparation & its subculture methods were used to determine the antimicrobial activity of the neem leaf extract. Different concentrations of neem leaf extracts in culture plates were used for the study. In this method inhibited zones were measured. RESULTS Neem leaf extracts showed considerable antimicrobial activity against four target pathogens. In agar well diffusion method it showed maximum antimicrobial activity against Enterococcus and Staphylococcus aureus. Growth of inhibition was increased as the concentration of the neem extract in agar media increased and was maximum for Staphylococcus aureus. CONCLUSIONS The extract of A. indica, has antimicrobial activity against target pathogens Enterococcus, Staphylococcus aureus, Pseudomonas, and E.coli. With the old medicinal knowledge on neem, better economic and therapeutic utilization can be done by using modern approaches of drug development. KEY WORDS Neem Leaf extract, Antimicrobial Activity, Agar Well Diffusion Test, Zone of Inhibition

Novel electronic biosensor for automated inoculum preparation to accelerate antimicrobial susceptibility testing

A key predictor of morbidity and mortality for patients with a bloodstream infection is time to appropriate antimicrobial therapy. Accelerating antimicrobial susceptibility testing from positive blood cultures is therefore key to improving patient outcomes, yet traditional laboratory approaches can require 2–4 days for actionable results. The eQUANT—a novel instrument utilizing electrical biosensors—produces a standardized inoculum equivalent to a 0.5 McFarland directly from positive blood cultures. This proof-of-concept study demonstrates that eQUANT inocula prepared from clinically significant species of Enterobacterales were comparable to 0.5 McF inocula generated from bacterial colonies in both CFU/ml concentration and performance in antimicrobial susceptibility testing, with ≥ 95% essential and categorical agreement for VITEK2 and disk diffusion. The eQUANT, combined with a rapid, direct from positive blood culture identification technique, can allow the clinical laboratory to begin antimicrobial susceptibility testing using a standardized inoculum approximately 2–3 h after a blood culture flags positive. This has the potential to improve clinical practice by accelerating conventional antimicrobial susceptibility testing and the resulting targeted antibiotic therapy.

Effect of melanin biosynthesis inhibition in the antifungal susceptibility of chromoblastomycosis agents

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by genera of melanized fungi: Fonsecaea, Cladophialophora, Phialophora, Exophiala and Rhinocladiella. Melanin is a virulence factor known to influence antifungal susceptibility. A specific inhibitor of melanin biosynthesis is tricyclazole. The aim of this study was to evaluate the effect of melanin inhibition on antifungal susceptibility of chromoblastomycosis agents and describe the susceptibility profile of some unusual CBM agents. Seventy-six clinical isolates, representing 13 species of the five main CBM agents genera, were studied. The antifungal susceptibility was performed according to the M38-A2 protocol of CLSI. In the melanin inhibition test, 16 mg/L of tricyclazole was added to the medium used in the inoculum preparation and the susceptibility assay. CBM agents were less susceptible to amphotericin B in comparison with azoles and terbinafine. The unusual species showed similar susceptibilities profiles to those of other species of the same genera. With tricyclazole exposition, MICs of terbinafine, posaconazole and itraconazole for Fonsecaea spp. significantly decreased (p<0,05). For Phialophora spp., this reduction was significative for posaconazole and itraconazole. For the other genera, there was a reduction in MICs of terbinafine and itraconazole, however, the statistical tests were not significant. Melanin inhibition can increase the antifungal susceptibility of most CBM agents to itraconazole and terbinafine, the main used drugs in the disease treatment. This increased susceptibility may open up new possibilities for therapy in refractory cases of CBM and/or caused by resistant fungal strains. Further studies are needed to confirm the same results in vivo.

Preparation of Komagataeibacter xylinus inoculum for bacterial cellulose biosynthesis using magnetically assisted external-loop airlift bioreactor

The aim of this study was to demonstrate the applicability of a novel magnetically-assisted external-loop airlift bioreactor (EL-ALB), equipped with RMF generators for the preparation of Komagataeibacter xylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200x higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible mechanical strength, nanostructure and total crystallinity index. The results obtained in this study may find multiple applications in any biotechnological processes requiring a high-quality bacterial inoculum.

Inoculum preparation conditions influence adherence of Salmonella enterica serovars on red leaf lettuce (Lactuca sativa)

Salmonella enterica has been increasingly linked to outbreaks involving consumption of fresh produce. While several studies have identified genes whose products are involved in mediating S. enterica -plant interactions, the use of varying experimental approaches, serovars and plant types have generated variable and conflicting data. The purpose of this study was to determine whether conditions under which inocula are prepared for in vitro plant interaction studies influence the outcome of these studies. Seven S. enterica serovars were grown in media of differing salinity and physical state, and incubated at either 25 or 37°C. These cultures were then used to inoculate red leaf lettuce and adherent microbes were subsequently recovered. While all serovars were influenced by inoculum preparation conditions, some showed greater variation than others. Analysis of pooled serovar data revealed that inocula prepared from either plates or biphasic cultures showed higher levels of interaction with red leaf lettuce than those prepared from broth cultures. Furthermore, incubation at 37°C enhanced adherence after 30s or 5d of contact time, while adherence levels after 1h of contact time were increased in low-salt media. Broth-grown inocula were highly influenced by medium salinity and incubation temperature, while plate- and biphasic-grown inocula were only minimally affected. Therefore, inocula prepared from bacteria grown on plates or in biphasic culture would be most suitable for studies aiming to evaluate strategies to interfere with plant- S. enterica interactions. However, pooled data mask serovar-specific responses, and care should be taken to extrapolate these findings to individual serovars. The previous association of a serovar with outbreaks involving leafy greens did not correlate with levels of interaction with red leaf lettuce, suggesting that the occurrence of these serovars in/on these commodities does not reflect their fitness in the plant environment.

Reliable and Standardized Animal Models to Study the Pathogenesis of Bluetongue and Schmallenberg Viruses in Ruminant Natural Host Species with Special Emphasis on Placental Crossing

Starting in 2006, bluetongue virus serotype 8 (BTV8) was responsible for a major epizootic in Western and Northern Europe. The magnitude and spread of the disease were surprisingly high and the control of BTV improved significantly with the marketing of BTV8 inactivated vaccines in 2008. During late summer of 2011, a first cluster of reduced milk yield, fever, and diarrhoea was reported in the Netherlands. Congenital malformations appeared in March 2012 and Schmallenberg virus (SBV) was identified, becoming one of the very few orthobunyaviruses distributed in Europe. At the start of both epizootics, little was known about the pathogenesis and epidemiology of these viruses in the European context and most assumptions were extrapolated based on other related viruses and/or other regions of the World. Standardized and repeatable models potentially mimicking clinical signs observed in the field are required to study the pathogenesis of these infections, and to clarify their ability to cross the placental barrier. This review presents some of the latest experimental designs for infectious disease challenges with BTV or SBV. Infectious doses, routes of infection, inoculum preparation, and origin are discussed. Particular emphasis is given to the placental crossing associated with these two viruses.