Evaluation of visible light and natural photosensitizers against Staphylococcus epidermidis and Staphylococcus saprophyticus planktonic cells and biofilm.

Antimicrobial photoinactivation (API) has shown some promise in potentially treating different nosocomial bacterial infections, however, its application on staphylococci, especially other than Staphylococcus aureus or methicillin-resistant S. aureus (MRSA) species is still limited. Although S. aureus is a well-known and important nosocomial pathogen, several other species of the genus, particularly coagulase-negative Staphylococcus (CNS) species such as Staphylococcus epidermidis and Staphylococcus saprophyticus, can also cause healthcare-associated infections and foodborne intoxications. CNS are often involved in resilient biofilm formation on medical devices and can cause infections in patients with compromised immune systems or those undergoing invasive procedures. In this study, the effects of chlorophyllin and riboflavin-mediated API on S. epidermidis and S. saprophyticus planktonic cells and biofilm are demonstrated for the first time. Based on the residual growth determination and metabolic reduction ability changes, higher inactivating efficiency of chlorophyllin-mediated API was determined against the planktonic cells of both tested species of bacteria and against S. saprophyticus biofilm. Some insights on whether aqueous solutions of riboflavin and chlorophyllin, when illuminated with optimal exciting wavelength (440 nm and 402 nm, respectively) generate O2-•, are also provided in this work.

Solid-State 13C Nuclear Magnetic Resonance Study of Soluble and Insoluble ß-Glucans Extracted from Candida lusitaniae.

ß-glucans are widely known for their biological activities. However, the choice of extraction method can significantly influence their structural characteristics, thereby potentially impacting their biological functions. In this paper, three fractions of ß-glucans were obtained from Candida lusitaniae yeast via alkali and hot-water extraction methods and were analyzed using solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Solid-state NMR spectroscopy was used as a nondestructive technique that preserves the structure of the analyzed molecules. The results suggest that differences in the ß-glucan structure are affected by the choice of extraction method. The main difference occurred in the 82-92 ppm region with signal presence suggesting that ß-glucans have a linear structure when hot-water-extracted, which is absent in alkali-extracted fractions resulting in the acquisition of ß-glucans with an ordered, possibly helical structure. A hot-water extracted water-insoluble (HWN) fraction consists of linear ß-1,3-glucans with other signals indicating the presence of ß-1,6-linked side chains, chitin and small amounts of α-glucan impurities. For those that are alkali-extracted, alkali-insoluble (AN) and water-soluble (AWS) fractions are structurally similar and consist of an ordered ß-1,3-glucan structure with ß-1,6-linked side chains and a significant amount of α-glucan and chitin in both fractions.

Novel leaderless bacteriocin geobacillin 6 from thermophilic bacterium Parageobacillus thermoglucosidasius.

Bacterial resistance to conventional antibiotics has urged us to develop alternative strategies against bacterial pathogens. Moreover, a demand for food products containing no chemical preservatives has led us to search for new alternative technologies for food preservation. Bacteriocins - ribosomally synthesized antimicrobial peptides - have been proposed as a new alternative to conventional antibiotics or chemicals for food preservation. This study describes biosynthesis and characterization of a novel leaderless bacteriocin, geobacillin 6, which was identified in the thermophilic bacterium Parageobacillus thermoglucosidasius. Its amino acid sequence shows low similarity to other bacteriocins and it is the first leaderless-type bacteriocin identified in thermophilic bacteria. Based on structure assessment, the bacteriocin forms a multi-helix bundle. Geobacillin 6 exhibits a relatively narrow antimicrobial spectrum, it is active in the µM range and against Gram-positive bacteria, mostly thermophilic species closely related to the producer strain. Bacteriocin demonstrates stability over pH 3-11 and is highly thermostable, retaining 100% of its activity after incubation at 95°C for 6 h. Geobacillin 6 has potential in the food industry and biotechnological processes where contamination with thermophilic bacteria is undesirable.

Investigation of amino acids related to Staphylococcus saprophyticus AG1 EstAG1 carboxylesterase catalytic function revealed a new family of bacterial lipolytic enzymes.

Most of the lipolytic enzymes (carboxylesterases, EC 3.1.1.1 and triacylglycerol acylhydrolases, EC 3.1.1.3) originate from bacteria and form a large group of functionally important enzymes that are also well known for their use in multiple biotechnology sectors. Rapid and increasing amount of bacterial lipolytic enzymes being discovered and characterized led to a necessity to classify them. More than twenty years ago bacterial lipolytic enzymes were originally classified into eight families and six true lipase sub-families based on the differences in their amino acid sequences and biochemical properties. Later, this classification was comprehensively updated to 19 families with eight subfamilies, and more recently, employing deeper comparative analysis methods, classification expanded to 35 families and 11 subfamilies. Bacterial lipolytic enzymes that cannot be classified into currently existing families are still being discovered. This work provides site-directed mutagenesis and differential scanning fluorimetry based investigation of catalytic function-related amino acids of previously discovered and characterized EstAG1 carboxylesterase from Staphylococcus saprophyticus AG1. Experimental results obtained in this work revealed that EstAG1 carboxylesterase can be placed into a new family of bacterial lipolytic enzymes.

Riboflavin- and chlorophyllin-based antimicrobial photoinactivation of Brevundimonas sp. ESA1 biofilms.

Some Brevundimonas spp. are globally emerging opportunistic pathogens that can be dangerous to individuals with underlying medical conditions and for those who are immunocompromised. Gram-negative Brevundimonas spp. can form resilient sessile biofilms and are found not only in different confined terrestrial settings (e.g., hospitals) but are also frequently detected in spacecraft which is inhabited by astronauts that can have altered immunity. Therefore, Brevundimonas spp. pose a serious health hazard in different environments, especially in its biofilm form. Conventional antimicrobials applied to disrupt, inactivate, or prevent biofilm formation have limited efficiency and applicability in different closed-loop systems. Therefore, new, effective, and safe biofilm control technologies are in high demand. The present work aimed to investigate antimicrobial photoinactivation (API) of Brevundimonas sp. ESA1 monocultural biofilms mediated by non-toxic, natural photosensitizers such as riboflavin (RF) and chlorophyllin (Chl) with an emphasis of this technology as an example to be safely used in closed-loop systems such as spacecraft. The present study showed that Chl-based API had a bactericidal effect on Brevundimonas sp. ESA1 biofilms at twice the lower irradiation doses than was needed when applying RF-based API. Long-term API based on RF and Chl using 450 nm low irradiance plate has also been studied in this work as a more practically applicable API method. The ability of Brevundimonas sp. ESA1 biofilms to reduce alamarBlue™ and regrowth analysis have revealed that after the applied photoinactivation, bacteria can enter a viable but non-culturable state with no ability to resuscitate in some cases.

FT-IR Method Limitations for ß-Glucan Analysis.

ß-glucans are known as biological response modifiers. However, different sources can result in structural differences and as a result differences in their biological activity. The hot water extraction method allows to obtain, high molecular weight ß-glucans without altering their structure by using strong chemicals, such as alkalis or acids. Analysis of ß-glucans by FT-IR and NMR spectroscopy in solid state is superior to analysis in solution as it allows researchers to study the preserved structure of the extracted polysaccharides. FT-IR spectroscopy was used in this study to make side-by-side comparison analysis of hot water extracted ß-glucans from different yeast sources. NMR spectroscopy was used to confirm findings made by FT-IR spectroscopy. Extracted ß-glucans exhibit characteristic structure of ß-1,3/1,6-linked glucans with noticeable levels of proteins, possibly in a form of oligopeptides, chitin and other impurities. ß-glucans obtained from C. guilliermondii, P. pastoris and S. pastorianus exhibited higher protein content. Differences in mannan, chitin and α-glucan content were also observed; however, the species-specific structure of obtained ß-glucans could not be confirmed without additional studies. Structural analysis of high molecular weight ß-glucans in solid state by FT-IR spectroscopy is difficult or limited due to band intensity changes and overlapping originating from different molecules.

Antimicrobial Photoinactivation Approach Based on Natural Agents for Control of Bacteria Biofilms in Spacecraft.

A spacecraft is a confined system that is inhabited by a changing microbial consortium, mostly originating from life-supporting devices, equipment collected in pre-flight conditions, and crewmembers. Continuous monitoring of the spacecraft's bioburden employing culture-based and molecular methods has shown the prevalence of various taxa, with human skin-associated microorganisms making a substantial contribution to the spacecraft microbiome. Microorganisms in spacecraft can prosper not only in planktonic growth mode but can also form more resilient biofilms that pose a higher risk to crewmembers' health and the material integrity of the spacecraft's equipment. Moreover, bacterial biofilms in space conditions are characterized by faster formation and acquisition of resistance to chemical and physical effects than under the same conditions on Earth, making most decontamination methods unsafe. There is currently no reported method available to combat biofilm formation in space effectively and safely. However, antibacterial photodynamic inactivation based on natural photosensitizers, which is reviewed in this work, seems to be a promising method.

Molecular Characterization of Uropathogenic Escherichia coli Reveals Emergence of Drug Resistant O15, O22 and O25 Serogroups.

Background and Objectives: Uropathogenic Escherichia coli (UPEC) are common pathogens causing urinary tract infections (UTIs). We aimed to investigate the relationship among clinical manifestation, serogroups, phylogenetic groups, and antimicrobial resistance among UPEC. Materials and Methods: One-hundred Escherichia coli isolates recovered from urine and ureteral scrapings were used for the study. The prevalence of antimicrobial resistance was determined by using European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. E. coli serogroups associated with UTI, as well as phylogenetic diversity were analyzed using multiplex PCR reactions. Results: Eighty-seven strains (87%) were isolated from females, while 13 (13%) from males. A high frequency of resistance to cephalosporins (43%) and fluoroquinolones (31%) was observed. Among UTI-associated serogroups O15 (32.8%), O22 (23.4%), and O25 (15.6%) were dominant and demonstrated elevated resistance rates. The E. coli phylogenetic group B2 was most common. These observations extended to pregnant patients with asymptomatic bacteriuria. Conclusions: Due to high rates of resistance, strategies using empirical therapy of second-generation cephalosporins and fluoroquinolones should be reconsidered in this population.

Geobacillin 26 - high molecular weight bacteriocin from a thermophilic bacterium.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

Atypical organic-solvent tolerant bacterial hormone sensitive lipase-like homologue EstAG1 from Staphylococcus saprophyticus AG1: Synthesis and characterization.

Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Some of the most industrially relevant enzymes of this type are lipolytic and their market is predicted to uphold leadership up till 2024. In this study, a novel bacterial hormone-sensitive lipase-like (bHSL) family homologue, designated EstAG1, was discovered by mining gDNA of bacteria isolated from fat contaminated soil in Lithuania. Putative lipolytic enzyme was cloned, overexpressed in E. coli, purified and characterized determining its biochemical properties. While the true physiological role of the discovered leaderless, ~36 kDa enzyme is unknown, metal-activated EstAG1 possessed optima at 45-47.5 °C, pH 7.5-8, with a generally intermediate activity profile between esterases and lipases. Furthermore, EstAG1 was hyperactivated by ethanol, dioxane and DMSO, implicating that it could be industrially applicable enzyme for the synthesis of valuable products such as biodiesel, flavor esters, etc. Sequence analysis and structure modeling revealed that the highest sequence homology of EstAG1 with the closest structurally and functionally described protein makes up only 26%. It was also revealed that EstAG1 has some differences in the bHSL family-characteristic conserved sequence motives. Therefore, EstAG1 presents interest both in terms of biotechnological applications and basic research.

Analysis of Aspergillus sp. lipase immobilization for the application in organic synthesis.

Nowadays, for the industrial implementations, especially in the area of organic synthesis, immobilized enzymes are preferred over their soluble forms. Present study aimed to find fast, cost-efficient, and effective way of lipase immobilization for the use in organic media. Lipase from Aspergillus sp. (Resinase A 2X) was immobilized utilizing cross-linking of enzyme aggregates, covalent immobilization on magnetite particles and adsorption-immobilization using pyrolyzed sugar industry waste product as a novel type of carrier. Covalently- and adsorption-immobilized preparations exhibited greater specific activities (5.61±0.18U/mg and 14.2±0.63U/mg, respectively) in organic reaction media than the soluble form of the enzyme (0.06±0.01U/mg). Enzyme immobilized on the sugar industry waste pyrolyis product was determined as a best way to hyperactivate Resinase A 2X and was chosen for the synthesis of flavor and fragrance compound 2-phenylethyl butanoate. Furthermore, in order to optimize 2-phenylethyl butanoate synthesis conditions, central compositional experimental plan was designed using RSM. It showed that in optimal reaction conditions (4.5h at 40.7°C, with 0.1M of substrate) conversion higher than 90% can be achieved. Studies of the operational stability showed enhanced reusability of adsorption-immobilized lipase (with each cycle, efficiency of the 2-PB synthesis diminished by 20-30%). The use of the sugar industry waste pyrolysis product as a carrier provides a novel, cheap, fast, cost-efficient and eco-friendly way of immobilization with some crucial points to be noted for the best productivity.

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study.

Electroporation is a phenomenon occurring due to exposure of cells to Pulsed Electric Fields (PEF) which leads to increase of membrane permeability. Electroporation is used in medicine, biotechnology, and food processing. Recently, as an alternative to electroporation by PEF, Pulsed ElectroMagnetic Fields (PEMF) application causing similar biological effects was suggested. Since induced electric field in PEMF however is 2-3 magnitudes lower than in PEF electroporation, the membrane permeabilization mechanism remains hypothetical. We have designed pilot experiments where Saccharomyces cerevisiae and Candida lusitaniae cells were subjected to single 100-250 µs electrical pulse of 800 V with and without concomitant delivery of magnetic pulse (3, 6 and 9 T). As expected, after the PEF pulses only the number of Propidium Iodide (PI) fluorescent cells has increased, indicative of membrane permeabilization. We further show that single sub-millisecond magnetic field pulse did not cause detectable poration of yeast. Concomitant exposure of cells to pulsed electric (PEF) and magnetic field (PMF) however resulted in the increased number PI fluorescent cells and reduced viability. Our results show increased membrane permeability by PEF when combined with magnetic field pulse, which can explain electroporation at considerably lower electric field strengths induced by PEMF compared to classical electroporation.

Lipase of Bacillus stratosphericus L1: Cloning, expression and characterization.

Present work was undertaken to discover new lipolytic enzymes as well as novel bacterial strains for applications in biotechnology. One of the isolated strains identified as Bacillus stratosphericus L1 produced extracellular lipase (LipBST) which was cloned and expressed in Escherichia coli. Purified mature enzyme had a molecular mass of 19kDa. Recombinant protein showed an activity of 6244.5U/mg at pH 9, 35°C. It was stable in the range of 35-55°C and retained more than 60% activity after incubation for 4h. LipBST was activated by organic solvents such as acetone and n-hexane. Lipase was inactivated by all investigated metal ions, inhibitors and detergents. LipBST was determined to be short-chain specific, but also hydrolyzed medium-, long-chain p-nitrophenyl and natural fatty substrates. The values of Vmax and KM for p-nitrophenyl butyrate, p-nitrophenyl caprylate, p-nitrophenyl decanoate were 1.1, 2.5, 0.1mMmin-1 and 5×10-2, 3.4×10-2, 194×10-2mM, respectively. Biochemical characteristics of LipBST suggest a great potential for various biotechnological applications including detergent formulation, bioremediation and organic synthesis processes.

High Performance Protein-Coated Microcrystals of Rhizomucor miehei Lipase: Preparation and Application for Organic Synthesis.

The goal of obtaining enzyme forms with higher catalytic activity, greater stability, and improved reusability has been pursued for the last few decades. Various novel biocatalyst designs have been created, and protein-coated microcrystals (PCMCs) are one of them. PCMC is an enzyme immobilization method based on simultaneous precipitation of protein and carrier, forming micron-sized enzyme-coated crystals. Highly active Rhizomucor miehei lipase (RML) PCMCs were prepared by immobilizing the protein onto K2SO4 as a carrier salt in acetone as a precipitating solvent. The formation of RML PCMCs was confirmed by scanning electron microscopy. Preparation of RML PCMCs was optimized by response surface methodology (RSM). Obtained PCMCs were found to be more active and stable during p-nitrophenyl palmitate hydrolysis in n-hexane, compared to liquid RML. The enzymatic activity and temperature optimum increased from 0.011 U/mg(soluble) lipase to 8.70 U/mg(immobilized) lipase and from 30 to 37 °C, respectively. Additionally, the ability of RML PCMCs to catalyze flavor ester 2-phenethyl octanoate synthesis was investigated. Some reaction parameters were optimized, resulting in 80 % conversion within 1 h with an enhanced reusability, compared to commercial immobilized RML preparation. Thus, PCMCs offer a cheap and effective technology for obtaining highly active lipase preparations for biocatalysis in organic media.

Biotransformation of eugenol via protocatechuic acid by thermophilic Geobacillus sp. AY 946034 strain.

The metabolic pathway of eugenol degradation by thermophilic Geobacillus sp. AY 946034 strain was analyzed based on the lack of data about eugenol degradation by thermophiles. TLC, GC-MS, and biotransformation with resting cells showed that eugenol was oxidized through coniferyl alcohol, and ferulic and vanillic acids to protocatechuic acid before the aromatic ring was cleaved. The cell-free extract of Geobacillus sp. AY 946034 strain grown on eugenol showed a high activity of eugenol hydroxylase, feruloyl-CoA synthetase, vanillate-O-demethylase, and protocatechuate 3,4-dioxygenase. The key enzyme, protocatechuate 3,4- dioxygenase, which plays a crucial role in the degradation of various aromatic compounds, was purified 135-fold to homogeneity with a 34% overall recovery from Geobacillus sp. AY 946034. The relative molecular mass of the native enzyme was about 450 ± 10 kDa and was composed of the non-identical subunits. The pH and temperature optima for enzyme activity were 8 and 60°C, respectively. The half-life of protocatechuate 3,4-dioxygenase at the optimum temperature was 50 min.