Important Microorganisms Research Articles

Fighting Staphylococcus epidermidis Biofilm-Associated Infections: Can Iron Be the Key to Success?

Staphylococcus epidermidis is one of the most important commensal microorganisms of human skin and mucosae. However, this bacterial species is also the cause of severe infections in immunocompromised patients, specially associated with the utilization of indwelling medical devices, that often serve as a scaffold for biofilm formation. S. epidermidis strains are often multidrug resistant and its association with biofilm formation makes these infections hard to treat. Their remarkable ability to form biofilms is widely regarded as its major pathogenic determinant. Although a significant amount of knowledge on its biofilm formation mechanisms has been achieved, we still do not understand how the species survives when exposed to the host harsh environment during invasion. A previous RNA-seq study highlighted that iron-metabolism associated genes were the most up-regulated bacterial genes upon contact with human blood, which suggested that iron acquisition plays an important role in S. epidermidis biofilm development and escape from the host innate immune system. In this perspective article, we review the available literature on the role of iron metabolism on S. epidermidis pathogenesis and propose that exploiting its dependence on iron could be pursued as a viable therapeutic alternative.

Utilization of Industrial Rosa damascena Mill. By-products and Cocoa Pod Husks as Natural Preservatives in Muffins

Cocoa Pod Husks (CPH) and by-product from supercritical CO2 extracted Rosa damascena Mill. (RDCO2) were used as biopreservatives in muffins. Both by-products were rich source of polyphenols: 28.3 ± 0.6 mg/g Dry Weight (DW) and 17.9 ± 0.7 mg/g DW RDCO2 and CPH, respectively, and exhibited potent antioxidant capacity: 449.1 ± 8.5 µmol Trolox Equivalents (TE)/g DW (by ORAC method) and 58.9 ± 2.1 µmol Gallic Acid Equivalents (GAE)/g DW (by HORAC method) for the RDCO2, and 373.8 ± 9.0 µmol TE/g DW (by ORAC) and 36.8 ± 3.8 µmol GAE/g DW (by HORAC) for the CPH. RDCO2 extracts successfully inhibited development of several important pathogenic and saprophytic microorganisms causing microbial spoilage of food systems. The control muffins were good for consumption up to the 17th day, while the products supplemented with RDCO2 and CPH: until 20th day of storage at 22 ± 0.5 °C. The amount of dietary fibers in muffins supplemented with both by-products increased 3 times (8.57 ± 0.12 %) compared to control (2.91 ± 0.12 %) and the polyphenolic compounds increased 2.5 times (from 50.0 ± 0.3 for the control to 185.9 ± 0.6 mg/g DW). For the first time by-product of supercritical CO2 extraction of Rosa damascena Mill. was characterized and used as natural and cheap biopreservative.

Identification <i>Escherichia coli</i> on some fisheries commodities in the traditional market of Mamuju, West Sulawesi

Fish is a source of high-quality protein. Protein in fish has a complete composition and amount of essential amino acids. However, fish is a type of food that is susceptible to biological damage and is susceptible to microbial contamination so that it quickly decays. One of the most important microorganisms in spoilage of fish meat is Escherichia coli bacteria. This study aims to determine the presence of Escherichia coli bacteria in scad, tuna and skipjack tuna. The research was carried out from July to September 2021. The research method used was a sample survey method, sampling was carried out at 3 market locations, namely the TPI market and the tasiu market. Furthermore, the samples were analyzed at the Laboratory of Fish Quarantine and Quality Control Center (BKIPM) Mamuju, West Sulawesi. The results showed that the largest E.coli content was found in tuna 1 in the TPI market, which was 20 MPN/g, and the smallest E.coli content was < 3 MPN/g, namely scad fish 1 in the tasiu and TPI markets, tuna 1 and 2 in the TPI market, tuna 2 in TPI market.

A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application.

Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (P tac or P trc ) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolR q cassette and the myo-inositol-inducible promoter P iolT1 . The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemA M and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

1,2-Propanediol production from glycerol via an endogenous pathway of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important microorganism and is used as a cell factory for many chemicals production. When glycerol was used as the carbon source, 1,3-propanediol was the main catabolite of this bacterium. K. pneumoniae ΔtpiA lost the activity of triosephosphate isomerase and prevented glycerol catabolism through the glycolysis pathway. But this strain still utilized glycerol, and 1,2-propanediol became the main catabolite. Key enzymes of 1,2-propanediol synthesis from glycerol were investigated in detail. dhaD and gldA encoded glycerol dehydrogenases were both responsible for the conversion of glycerol to dihydroxyacetone, but overexpression of the two enzymes resulted in a decrease of 1,2-propanediol production. There are two dihydroxyacetone kinases (I and II), but the dihydroxyacetone kinase I had no contribution to dihydroxyacetone phosphate formation. Dihydroxyacetone phosphate was converted to methylglyoxal, and methylglyoxal was then reduced to lactaldehyde or hydroxyacetone and further reduced to form 1,2-propanediol. Individual overexpression of mgsA, yqhD, and fucO resulted in increased production of 1,2-propanediol, but only the combined expression of mgsA and yqhD showed a positive effect on 1,2-propanediol production. The process parameters for 1,2-propanediol production by Kp ΔtpiA-mgsA-yqhD were optimized, with pH 7.0 and agitation rate of 350rpm found to be optimal. In the fed-batch fermentation, 9.3g/L of 1,2-propanediol was produced after 144h of cultivation, and the substrate conversion ratio was 0.2g/g. This study provides an efficient way of 1,2-propanediol production from glycerol via an endogenous pathway of K. pneumoniae.Key points• 1,2-Propanediol was synthesis from glycerol by a tpiA knocked out K. pneumoniae• Overexpression of mgsA, yqhD, or fucO promote 1,2-propanediol production• 9.3g/L of 1,2-propanediol was produced in fed-batch fermentation.

Ammonia-oxidizing bacteria rather than ammonia-oxidizing archaea dominate nitrification in a nitrogen-fertilized calcareous soil

Microbe-driven nitrification is a key process that affects nitrogen (N) utilization by plants and N loss to the environment in agro-ecosystems. Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are important microorganisms that dominate the ammonia oxidation process (the first and rate-limiting step of nitrification). Calcareous soils are widely distributed, accounting for more than 30% of the Earth's land. However, the effects of long-term N fertilization on the potential nitrification rate (PNR) and on AOA and AOB in calcareous soils are poorly understood. In this study, we comprehensively assessed the effects of N application (applied at five rates as urea with 0, 73.5, 105, 136.5 and 250 kg N ha−1 for 12 years) on soil chemical characteristics, PNR, N use efficiency (NUE) and the community characteristics of AOB and AOA in a calcareous soil. N application rate affected AOB beta diversity more than that of AOA. Compared to no N control, N application significantly decreased the relative abundance of Group I.1b clade A of AOA and Nitrosospira cluster 3a.2 of AOB, but increased Nitrosomonas cluster 7 of AOB. The relative abundance of Nitrosospira cluster 3a.2 of AOB was negatively correlated with PNR. A structural equation model showed a direct effect of N application rate on the content of soil organic matter and nitrate, the alpha and beta diversity of AOA and AOB. Nitrate and AOB beta diversity were the key factors affecting PNR. Overall, the alpha, beta diversity and community composition of AOB contribute more to PNR than AOA in calcareous soils with high organic matter content. Understanding the relationship between the characteristics of AOA and AOB in calcareous soils and PNR will help to improve NUE.

Hand Hygiene Evaluation Using Two Different Evaluation Tools and Hand Contamination of Veterinary Healthcare Workers in a Swiss Companion Animal Clinic.

Hand hygiene (HH) is the most important measure to prevent nosocomial infections. HH compliance in companion animal clinics has been reported to be poor. The present study compared an online application with the WHO evaluation form to assess the WHO five moments of HH in a Swiss companion animal clinic. In 202 hand swabs from 87 staff members, total viable count (TVC) before and after patient contact was evaluated and the swabs were tested for selected antimicrobial resistant microorganisms of public health importance. HH compliance (95% confidence interval) was 36.6% (33.8–39.5%) and was similar when assessed with the two evaluation tools. HH differed between hospital areas (p = 0.0035) and HH indications (p < 0.0001). Gloves were worn in 22.0% (18.0–26.6%) of HH observations and were indicated in 37.2% (27.3–48.3%) of these observations. Mean TVC before patient contact was lower (0.52 log CFU/cm2) than after patient contact (1.02 log CFU/cm2) but was similar before patient contact on gloved and ungloved hands. Three hand swabs (1.5% (0.4–4.3%)) were positive for methicillin-resistant Staphylococcus aureus. Gloving should not be regarded as a substitute for HH. Overall, HH in companion animal medicine should urgently be fostered.

Review of microbial touchscreen contamination for the determination of reasonable ultraviolet disinfection doses.

Background: Touchscreens are usually microbially contaminated and can therefore act as fomites inside and outside healthcare environments. Due to the increasing use of such touchscreens and the growing awareness of infection risks, approaches that allow safe and automatic disinfection are desired. Ultraviolet (UV) irradiation, with its known antimicrobial efficacy, could achieve this goal, but should be executed with limited touchscreen degradation, disinfection duration, and energy consumption. It should also pose as little harm as possible to humans even in case of failure. Materials and methods: A literature search was performed first to identify the microorganisms most commonly found on touchscreens. Then, the 90% reduction doses (D90 doses) for the different relevant microorganisms and UV spectral ranges were determined from the literature, and irradiation doses are suggested that should reduce most of these important microorganisms by 5 log-levels. Results: The most frequent microorganisms are staphylococci, bacilli, micrococci, enterococci, pseudomonads and E. coli with small differences between hospital and community environments, if antibiotic resistance properties are ignored. The determined irradiation doses for a 5 log-reduction of the most frequent microorganisms are about 40 mJ/cm2, 80 J/cm2, 500 J/cm2 and 50 mJ/cm2 for the UV spectral ranges UVC, UVB, UVA and far-UVC, respectively. These doses are also sufficient to inactivate all nosocomial ESKAPE pathogens on touchscreens by at least 99.999%.Conclusion: Disinfection is achievable in all UV spectral ranges, with UVC being the most effective, enabling automatic disinfection within a minute or less. The much higher doses required in the UVB and UVA spectral range result in much longer disinfection durations, with the advantage of a reduced risk to humans. For all kinds of UV irradiation, the doses should be limited to reasonable values to avoid irradiating an already more or less sterile surface and to prevent degradation of touchscreen devices.

Isolation and identification of bacteria associated with fermentation of melon seed for Ogiri Sold in Owerri, Nigeria

In order to ascertain the public health implication of the consumption of the fermented melon (Cucumismelo), known as Ogiri, consumed in all South Eastern Nigeria and beyond as a food condiment, thirty samples of locally fermented melon seeds, Ogiri, were randomly purchased from three markets in Owerri metropolis in Imo State. These samples were microbiologically analysed using pour plate technique on nutrient agar, MacConkey and Salmonella Shigella agar at 370C for 24 hours. The viable and mean counts were determined and the data obtained were statistically analyzed. There was no significant difference between the contamination of the samples (p&gt;0.05). These organisms, following the order of predominance were isolated; Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The presence of these microorganisms of public health importance in food condiments pose a risk of enteritis and other food borne diseases in some individuals after its intake. Thus, the health organizations should embark on public and personal enlightenment programs targeted at both the producer and the consumer.

Recombinant and endogenous ways to produce methylated phospholipids in Escherichia coli

Escherichia coli is the daily workhorse in molecular biology research labs and an important platform microorganism in white biotechnology. Its cytoplasmic membrane is primarily composed of the phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). As in most other bacteria, the typical eukaryotic phosphatidylcholine (PC) is not a regular component of the E. coli membrane. PC is known to act as a substrate in various metabolic or catabolic reactions, to affect protein folding and membrane insertion, and to activate proteins that originate from eukaryotic environments. Options to manipulate the E. coli membrane to include non-native lipids such as PC might make it an even more powerful and versatile tool for biotechnology and protein biochemistry. This article outlines different strategies how E. coli can be engineered to produce PC and other methylated PE derivatives. Several of these approaches rely on the ectopic expression of genes from natural PC-producing organisms. These include PC synthases, lysolipid acyltransferases, and several phospholipid N-methyltransferases with diverse substrate and product preferences. In addition, we show that E. coli has the capacity to produce PC by its own enzyme repertoire provided that appropriate precursors are supplied. Screening of the E. coli Keio knockout collection revealed the lysophospholipid transporter LplT to be responsible for the uptake of lyso-PC, which is then further acylated to PC by the acyltransferase-acyl carrier protein synthetase Aas. Overall, our study shows that the membrane composition of the most routinely used model bacterium can readily be tailored on demand.Key points• Escherichia coli can be engineered to produce non-native methylated PE derivatives.• These lipids can be produced by foreign and endogenous proteins.• Modification of E. coli membrane offers potential for biotechnology and research.Graphical abstract

Structure and Topology Prediction of Phage Adhesion Devices Using AlphaFold2: The Case of Two Oenococcus oeni Phages.

Lactic acid bacteria (LAB) are important microorganisms in food fermentation. In the food industry, bacteriophages (phages or bacterial viruses) may cause the disruption of LAB-dependent processes with product inconsistencies and economic losses. LAB phages use diverse adhesion devices to infect their host, yet the overall picture of host-binding mechanisms remains incomplete. Here, we aimed to determine the structure and topology of the adhesion devices of two lytic siphophages, OE33PA and Vinitor162, infecting the wine bacteria Oenococcus oeni. These phages possess adhesion devices with a distinct composition and morphology and likely use different infection mechanisms. We primarily used AlphaFold2, an algorithm that can predict protein structure with unprecedented accuracy, to obtain a 3D model of the adhesion devices’ components. Using our prior knowledge of the architecture of the LAB phage host-binding machineries, we also reconstituted the topology of OE33PA and Vinitor162 adhesion devices. While OE33PA exhibits original structures in the assembly of its bulky adhesion device, Vinitor162 harbors several carbohydrate-binding modules throughout its long and extended adhesion device. Overall, these results highlight the ability of AlphaFold2 to predict protein structures and illustrate its great potential in the study of phage structures and host-binding mechanisms.

Update on Dihydropteroate Synthase (DHPS) Mutations in Pneumocystis jirovecii.

A Pneumocystis jirovecii is one of the most important microorganisms that cause pneumonia in immunosupressed individuals. The guideline for treatment and prophylaxis of Pneumocystis pneumonia (PcP) is the use of a combination of sulfa drug-containing trimethroprim and sulfamethoxazole. In the absence of a reliable method to culture Pneumocystis, molecular techniques have been developed to detect mutations in the dihydropteroate synthase gene, the target of sulfa drugs, where mutations are related to sulfa resistance in other microorganisms. The presence of dihydropteroate synthase (DHPS) mutations has been described at codon 55 and 57 and found almost around the world. In the current work, we analyzed the most common methods to identify these mutations, their geographical distribution around the world, and their clinical implications. In addition, we describe new emerging DHPS mutations. Other aspects, such as the possibility of transmitting Pneumocystis mutated organisms between susceptible patients is also described, as well as a brief summary of approaches to study these mutations in a heterologous expression system.

CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum

Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.

The role of topic vaginal products as part of the therapeutic strategy in uterine cervix lesions

It has been widely demonstrated so far that lactobacilli represent probably the most important microorganisms which are developed at the level of the vaginal cavity and which are responsible for the maintenance of an adequate environment and pH at this level. Once the number of these microorganisms decrease the pH value increase leading to the apparition of a basic medium, which favors the development of certain microorganisms such as human papilloma virus. Meanwhile it is widely known that the presence of this virus is associated with an increased risk of developing premalignant and even malignant conditions. The aim of the current paper is to investigate the correlation between the vaginal pH, the risk of human papilloma virus infection and, in the meantime, to explore the possible benefits of local application of Cerviron, a topic, vaginal product which seems to significantly influence the risk of cervical lesions development.

The Recent Trend in the Use of Multistrain Probiotics in Livestock Production: An Overview.

Simple SummaryProbiotics are live microorganisms that confer a health benefit to the administered animal when ingested. Their use has been an effective alternative to antimicrobial growth promoters in the livestock industry, and they could be bacteria or non-bacterial species or strains. However, there has been an increasing trend in administering multistrain probiotics in recent years. Multistrain probiotics comprise two or more species or strains of important microorganisms as a consortium beneficial to the administered animal. Several studies are being carried out to explore their potency or efficiency. They have proven to be a promising alternative to antibiotics growth promoters and were responsible for enhancing gut health, growth performance, maintaining a balance in gut microbiota, stimulating immunity against pathogenic organisms, improving digestion, and overall production efficiency in ruminants, poultry, and swine production.It has been established that introducing feed additives to livestock, either nutritional or non-nutritional, is beneficial in manipulating the microbial ecosystem to maintain a balance in the gut microbes and thereby improving nutrient utilization, productivity, and health status of animals. Probiotic use has gained popularity in the livestock industry, especially since antimicrobial growth promoter’s use has been restricted due to the challenge of antibiotic resistance in both animals and consumers of animal products. Their usage has been linked to intestinal microbial balance and improved performance in administered animals. Even though monostrain probiotics could be beneficial, multistrain probiotics containing two or more species or strains have gained considerable attention. Combining different strains has presumably achieved several health benefits over single strains due to individual isolates’ addition and positive synergistic adhesion effects on animal health and performance. However, there has been inconsistency in the effects of the probiotic complexes in literature. This review discusses multistrain probiotics, summarizes selected literature on their effects on ruminants, poultry, and swine productivity and the various modes by which they function.

Metabolic profiling, ADME pharmacokinetics, molecular docking studies and antibacterial potential of Phyllantus muellerianus leaves

Global increase in the level of antimicrobial resistance among bacterial pathogens has prompted the search for alternative treatment from medicinal plants. Phyllantus muellerianus leaves has been used traditionally against microorganisms of medical importance, hence the need to evaluate the pharmacological pathways and mode of actions using in vitro and in silico approaches. Clinical isolates of eight (8) microorganisms associated with urinary tract infections were obtained and identified using morphological and biochemical methods. Phyllantus muellerianus leaves were extracted and purified by solvent partitioning. Ethyl acetate fraction of PM had the highest yield and zone diameter range from 13.5 ± 1.00 to 28 ± 1.53 mm. The rate of protein leakage per time interval of Staphylococcus aureus increased from 9.29 μg/ml at 0 min to 17.43 μg/ml at 120 min while leakage in Candida albicans also increased from 8.57 μg/ml at 0 min to 70.43 μg/ml at 120 min. GCMS fingerprints, pharmacodynamics and pharmacokinetic studies revealed the active agent as quindoline, an azaindole and isotere of indoles having a binding energy of −9.1 kcal/mol. Analyses of the structural and atomic orientations of quindoline, and superimposition on ciprofloxacin, a common antibiotic revealed an interesting comparison, effecting a stronger binding affinity of Quindoline-HMG-CoA complex.

Microbial community of soda Lake Van as obtained from direct and enriched water, sediment and fish samples

Soda lakes are saline and alkaline ecosystems that are considered to have existed since the first geological records of the world. These lakes support the growth of ecologically and economically important microorganisms due to their unique geochemistry. Microbiota members of lakes are valuable models to study the link between community structure and abiotic parameters such as pH and salinity. Lake Van is the largest endroheic lake and in this study, bacterial diversity of lake water, sediment, and pearl mullet (inci kefali; Alburnus tarichi), an endemic species of fish which are collected from different points of the lake, are studied directly and investigated meticulously using a metabarcoding approach after pre-enrichment. Bacterial community structures were identified using Next Generation Sequencing of the 16S rRNA gene. The analysis revealed that the samples of Lake Van contain high level of bacterial diversity. Direct water samples were dominated by Proteobacteria, Cyanobacteria, and Bacteroidota, on the other hand, pre-enriched water samples were dominated by Proteobacteria and Firmicutes at phylum-level. In direct sediment samples Proteobacteria, whereas in pre-enriched sediment samples Firmicutes and Proteobacteria were determined at highest level. Pre-enriched fish samples were dominated by Proteobacteria and Firmicutes at phylum-level. In this study, microbiota members of Lake Van were identified by taxonomic analysis.

IN VITRO ANTIMICROBIAL ACTIVITY OF CARVACROL AGAINST SHRIMP PATHOGENS AND ITS USE AS FEED ADDITIVE FOR THE PACIFIC WHITE SHRIMP

This study aimed to evaluate the in vitro effect of carvacrol on different microorganisms of importance in shrimp farming, as well as its in vivo effect on zootechnical, immunological and microbiological performance, as well as resistance, of Litopenaeus vannamei challenged with Vibrio parahaemolyticus. In particular, the antimicrobial activity of carvacrol was evaluated in vitro by analysis of the minimum inhibitory concentration (MIC) and by agar diffusion disc with Gram-negative and Gram-positive bacteria. The in vivo experiment was conducted using different concentrations of carvacrol (1, 3, 4 and 6 mg mL-1) added to shrimp feed, together with a control diet without carvacrol. After four weeks, zootechnical, immunological and microbiological parameters, as well as resistance, of animals challenged with V. parahaemolyticus were evaluated. The MIC of Vibrio alginolyticus and Vibrio harveyi was 0.078 mg mL-1, while for the other bacteria, it was 0.156 mg mL-1 of carvacrol. The greatest halos of inhibition were observed in V. parahaemolyticus and Vibrio harveyi with significant differences demonstrated for the other microorganisms, except Escherichia coli. The in vivo results showed no significant differences among treatments. In conclusion, the antimicrobial activity of carvacrol was confirmed with Gram-negative and Gram-positive bacteria, and it is suggested that its antimicrobial potential is more effective against Vibrio spp. However, the concentrations of carvacrol used in vivo did not affect the parameters evaluated.

Antibiofilm and cytotoxic effect of 3,3′-dihydroxycurcumin (DHC) as photosensitizer agent in antimicrobial photodynamic therapy for endodontic purposes

BackgroundCurcuminoids have been designed not only to improve chemical and metabolic stability of curcumin (CUR), but also to increase its antimicrobial activity, without effecting its ability as photosensitizer agent in antimicrobial photodynamic therapy (aPDT) with light emitting diode (LED). This study evaluated the antimicrobial and antibiofilm action of curcumin analog DHC (or 3,3′-dihydroxycurcumin), submitted or not to LED irradiation, on microorganisms of endodontic importance and its influence on fibroblasts viability. MethodsDHC was synthetized by modified Pablon's methodology and the experiments were conducted under irradiation or not with indium gallium nitride-based LED (440-480nm, 100 mW/cm2, 0.78 cm2,60 s). The antimicrobial activity of CUR and DHC were determined by the Minimum Inhibitory and Bactericidal Concentration assays against Gram-positive and Gram-negative bacteria and the effect of both compounds on fibroblast viability was tested using colorimetric assays. They were also evaluated on 72h and 7days single-species biofilms and on 14 days multispecies biofilms formed inside dentin tubules by bacterial colonies counts and confocal microscopy, respectively. Data were analyzed statistically considering p<0.05. ResultsDHC had bactericidal effect against all bacteria tested higher than CUR, in planktonic conditions. CUR and DHC (at 39 and 19 μg/mL, respectively) were cytocompatible and LED irradiation reduced fibroblast viability, regardless of compound. CUR and DHC reduced the growth of single-species biofilms and the effect of aPDT was bacteria dependent. DHC reduced more than 70% of microorganisms from multispecies biofilms, superior to CUR effect. ConclusionsDHC showed low cytotoxicity and antibiofilm effect similar to curcumin, when submitted or not to aPDT, and could be further explored as a bioactive compound for endodontic purposes.

The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

BackgroundBacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP.ResultsAs proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity.ConclusionOur results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level.Graphic abstract