Optimization of Growing Medium and Preservation Methods for Plant Beneficial Bacteria, and Formulating a Microbial Biopreparation for Raspberry Naturalization

The current study focuses on the optimization of bacterial growing medium composition, including the carbon and nitrogen source in different concentrations, the pH value of the medium and the temperature. Optimization was performed for four environmental bacterial isolates belonging to the genera Arthrobacter, Pseudomonas and Rhodococcus, which were previously obtained from wild raspberries. These bacteria proved to be potent antagonists against certain fungal and fungal-like plant pathogens. Furthermore, three preservation methods and three sample preparation techniques were evaluated. In addition, a prebiotic supplementary blend based on previous research was tested. The research included a pot experiment to estimate the influence of bacterial cultures on the growth of plant shoots and roots, on the soil enzymatic activity and the content of macronutrients, minerals and nitrogen in the soil depending on the naturalization strategy. The best carbon and nitrogen source were chosen. The addition of a supplementary blend resulted in the increased growth of two bacterial isolates. Bacterial inoculum applied to the roots and watering resulted in increased shoot mass in objects infected with plant pathogens, although in plants without the pathogen infection, bacterial inoculum resulted in the decreased mass of plants. Naturalization strategy should be matched to the pathogens present at plantations.

Critical Factors for Optimum Biodegradation of Bast Fiber’s Gums in Bacterial Retting

Bast fiber plants require a post-harvest process to yield useable natural cellulosic fibers, denoted as retting or degumming. It encompasses the degradation of the cell wall’s non-cellulosic gummy substances (NCGs), facilitating fibers separations, setting the fiber’s quality, and determining downstream usages. Due to the inconvenience of traditional retting practices, bacterial inoculum and enzyme applications for retting gained attention. Therefore, concurrent changes of agroclimatic and socioeconomic conditions, the conventional water retting confront multiple difficulties, bast industries become vulnerable, and bacterial agents mediated augmented bio-retting processes trying to adapt to sustainability. However, this process’s success demands a delicate balance among substrates and retting-related biotic and abiotic factors. These critical factors were coupled to degrade bast fibers NCGs in bacterial retting while holistically disregarded in basic research. In this study, a set of factors were defined that critically regulates the process and requires to be comprehended to achieve optimum retting without failure. This review presents the bacterial strain characteristics, enzyme potentials, specific bast plant cell wall’s structure, compositions, solvents, and interactions relating to the maximum NCGs removal. Among plants, associated factors pectin is the primary biding material that determines the process’s dynamics, while its degree of esterification has a proficient effect through bacterial enzymatic degradation. The accomplished bast plant cell wall’s structure, macerating solvents pH, and temperature greatly influence the bacterial retting process. This article also highlights the remediation process of water retting pollution in a biocompatible manner concerning the bast fiber industry’s endurance.

Central Composite Design: a Response Surface Methodology Approach in Biodegradation of Textile Dye Wastewater

Objectives : This study evaluated and identified the removal of colors and chemocal oxygen demands from thextile dye effluents by Bacillus cereus isolated from the local textile wastewater treatment plant.Methods : Central composite design (CCD) from response surface methodology (RSM) was applied in order to achieve the optimized treatment process condition for the textile dyes wastewater degradation. Two-level of three process parameters with six center points resulted a total of twenty runs of experiments were performed. Bacterial inoculum (-1,+1) (%, v/v), agitation (-1, +1) (rpm), and pH (-1, +1) were tested.Results and Discussion : During the ten days of biodegradation process, highest decolourization achieved was 88.67% with low pH and agitation; and medium level of initial concentration of bacterial inoculum. Highest chemical oxygen demand (COD) removal was achieved with 99.20% from high pH (pH 10), low agitation (100 rpm) and high initial concentration of bacterial inoculum (15%, v/v).Conclusions : The biological treatments was able to remove colour and chemical oxygen demand with application of CCD, giving the optimum settings of the three process parameters studied.

Microscopic Analysis of Bacterial Inoculum Effect Using Micropatterned Biochip

Antimicrobial resistance has become a major problem in public health and clinical environments. Against this background, antibiotic susceptibility testing (AST) has become necessary to cure diseases in an appropriate and timely manner as it indicates the necessary concentration of antibiotics. Recently, microfluidic based rapid AST methods using microscopic analysis have been shown to reduce the time needed for the determination of the proper antibiotics. However, owing to the inoculum effect, the accurate measurement of the minimal inhibitory concentration (MIC) is difficult. We tested four standard bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, against five different antibiotics: piperacillin, cefotaxime, amikacin, levofloxacin, and ampicillin. The results showed that overall, the microfluidic system has a similar inoculum effect compared to the conventional AST method. However, due to the different testing conditions and determination protocols of the growth of the microfluidic based rapid AST, a few results are not identical to the conventional methods using optical density. This result suggests that microfluidic based rapid AST methods require further research on the inoculum effect for practical use in hospitals and can then be used for effective antibiotic prescriptions.

Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology

Background Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria–bacteriophage co-cultures, are required. Results In the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage–bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13-fold increase). Conclusions Therefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.

Evaluation of ebselen in resolving a methicillin-resistant Staphylococcus aureus infection of pressure ulcers in obese and diabetic mice

Pressure ulcers (PUs) are a source of morbidity in individuals with restricted mobility including individuals that are obese or diabetic. Infection of PUs with pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), impairs ulcers from healing. The present study evaluated ebselen as a topical antibacterial to treat MRSA-infected PUs. Against two different S. aureus strains, including MRSA USA300, resistance to ebselen did not emerge after 14 consecutive passages. Resistance to mupirocin emerged after only five passages. Additionally, ebselen was found to exert a modest postantibiotic effect of five hours against two MRSA strains. Ebselen was subsequently evaluated in MRSA-infected PUs in two models using obese and diabetic mice. In obese mice, topical ebselen (89.2% reduction) and oral linezolid (84.5% reduction) similarly reduced the burden of MRSA in infected PUs. However, in diabetic mice, topical ebselen (45.8% reduction in MRSA burden) was less effective. Histopathological evaluation of ulcers in diabetic mice determined that ebselen treatment resulted in fewer bacterial colonies deep within the dermis and that the treatment exhibited evidence of epithelial regeneration. Topical mupirocin was superior to ebselen in reducing MRSA burden in infected PUs both in obese (98.7% reduction) and diabetic (99.3% reduction) mice. Ebselen’s antibacterial activity was negatively impacted as the bacterial inoculum was increased from 105 CFU/mL to 107 CFU/mL. These results suggest that a higher dose of ebselen, or a longer course of treatment, may be needed to achieve a similar effect as mupirocin in topically treating MRSA-infected pressure ulcers.

Dynamic interactions between cephalexin and macrophages on different Staphylococcus aureus inoculum sizes: a tripartite in vitro model

Background The bactericidal activity of an antimicrobial drug is generally assessed by in vitro bacterial time-kill experiments which do not include any components of the immune system, even though the innate immunity, the primary host defence, is probably able to kill a large proportion of pathogenic bacteria in immunocompetent patients. We developed an in vitro tripartite model to investigate the joint action of C57Bl/6 murine bone-marrow-derived macrophages and cephalexin on the killing of Staphylococcus aureus. Results By assessing the bactericidal effects on four bacterial inoculum sizes, we showed that macrophages can cooperate with cephalexin on inoculum sizes lower than 106 CFU/mL and conversely, protect S. aureus from cephalexin killing activity at the highest inoculum size. Cell analysis by flow cytometry revealed that macrophages were rapidly overwhelmed when exposed to large inoculums. Increasing the initial inoculum size from 105 to 107 CFU/mL increased macrophage death and decreased their ability to kill bacteria from six hours after exposure to bacteria. The addition of cephalexin at 16-fold MIC to 105 and 106 CFU/mL inoculums allowed the macrophages to survive and to maintain their bactericidal activity as if they were exposed to a small bacterial inoculum. However, with the highest inoculum size of 107 CFU/mL, the final bacterial counts in the supernatant were higher with macrophages plus cephalexin than with cephalexin alone. Conclusions These results suggest that if the bacterial population at the infectious site is low, as potentially encountered in the early stage of infection or at the end of an antimicrobial treatment, the observed cooperation between macrophages and cephalexin could facilitate its control.

Strategy for Mass Production of Lytic Staphylococcus Aureus Bacteriophage pSa-3: Contribution of Multiplicity of Infection

BackgroundAntibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures are required.ResultsIn the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13 fold increase).ConclusionsTherefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, which this combinatorial strategy could decrease phage production time. Further, we suggest that response surface methodology has the potential for optimizing mass production of lytic bacteriophages.

Metagenomics Analysis Reveals An Extraordinary Bacterial Diversity in Anisakids (Nematoda: Anisakidae) L3 Larvae

Background: L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as agents causing allergies and as potential vectors of pathogens microrganisms. In spite of the close bacteria-nematode relationship very little is known of the Anisakids microbiote. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of Anisakids when the L3 larvae migrate through the muscles. As a consequence the bacterial inoculum will be spread, affecting the quality of the fish, and possible clinical effects cannot be discardedResults: A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 area were studied. Bacteria were taxonomically characterized through 1803 representative OTUsequences. Fourteen Phyla, 31 Classes, 52 Orders, 129 Families and 187 genera were unambiguously identified. We have found as part of microbiome an average of 123 OTUs per L3 individual. Diversity Indices (Shannon and Simpson indices) indicate an extraordinary diversity of bacteria at a OTU level. There are clusters of Anisakids individuals (samples) defined by the associated bacteria which however are not significantly related with fish hosts or anisakid taxa. This suggests that association or relationship among bacteria in Anisakids, exists without the influence of fishes or nematodes. The lack of relationships with hosts of Anisakids taxa has to be necessarily expresssed by the association among bacterial OTUs or other taxonomical levels which are ranging from OTUs to phylum level. Conclusions: There are significant biological structural associations of microbiota in anisakid nematodes which manifest in clusters of bacteria ranging from plyllum to genus level which could also be anindicator of degree of fish contamination or from the geographic zone of fish capture. Actinobacteria, Aquificae, Firmicutes, and Proteobacteria are the phyla whose abundance value discriminate for difining such structures.

First Report on Infection of Eucalyptus pellita Seeds by Ralstonia solanacearum

Bacterial wilt is one of major threats to eucalyptus plantations which may cause significant losses. Until now, study about bacterial wilt on Eucalyptus pellita in Indonesia has been very limited, especially about the presence of the pathogen on or in the seeds. This study aims to provide evidence of the existence of the R. solanacearum bacterium on or in E. pellita seeds. Detection of seed-borne bacteria is determined by several approaches such as (i) direct detection using universal and selective media in the laboratory, (ii) the nursery test, and (iii) species-specific molecular detection. The results of our study indicate that R. solanacearum can be detected from eucalyptus seeds using universal and selective media in the laboratory, nursery test, and molecular-based detection using the Enrichment PCR method. The bacterial inoculum is also proven to exist both on the surface of and inside the eucalyptus seeds. This is the first report that R. solanacearum is a seed-borne pathogen in E. pellita seeds. Previous studies in different agricultural systems show that the effective method used to control the pathogen is through seed treatments using biological, physical, and chemical approaches.