Morpholinium-based Ionic Liquids as Potent Antibiofilm and Sensitizing Agents for the Control of Pseudomonas aeruginosa.

Rising antimicrobial resistance is a critical threat to worldwide public health. To address the increasing antibiotic tolerance, diverse antimicrobial agents are examined for their ability to decrease bacterial resistance. One of the most relevant and persistent human pathogens is Pseudomonas aeruginosa. Our study investigates the anti-biofilm and sensitizing activity of 12 morpholinium-based ionic liquids with herbicidal anions on four clinically relevant P. aeruginosa strains. Among all tested compounds, four ionic liquids prevented biofilm formation at sub-minimum inhibitory concentrations for all investigated strains. For the first time, we established a hormetic effect on biofilm formation for P. aeruginosa strains subjected to an ionic liquid treatment. Interestingly, while ionic liquids with 4,4-didecylmorpholinium [Dec2Mor]+ are more efficient against planktonic bacteria, 4-decyl-4-ethylmorpholinium [DecEtMor]+ showed more potent inhibition of biofilm formation. Ionic liquids with 4,4-didecylmorpholinium ([Dec2Mor]+) cations even induced biofilm formation by strain 39016 at high concentrations due to flocculation. Morpholinium-based ionic liquids were also shown to enhance the efficacy of commonly used antibiotics from different chemical groups. We demonstrate that this synergy is associated with the mode of action of the antibiotics.

Antimicrobial and Virucidal Potential of Morpholinium-Based Ionic Liquids.

Witnessed by the ongoing spread of antimicrobial resistant bacteria as well as the recent global pandemic of the SARS-CoV-2 virus, the development of new disinfection strategies is of great importance, and novel substance classes as effective antimicrobials and virucides are urgently needed. Ionic liquids (ILs), low-melting salts, have been already recognized as efficient antimicrobial agents with prospects for antiviral potential. In this study, we examined the antiviral activity of 12 morpholinium based herbicidal ionic liquids with a tripartite test system, including enzyme inhibition tests, virucidal activity determination against five model viruses and activity against five bacterial species. The antimicrobial and enzymatic tests confirmed that the inhibiting activity of ILs corresponds with the number of long alkyl side chains and that [Dec2Mor]+ based ILs are promising candidates as novel antimicrobials. The virucidal tests showed that ILs antiviral activity depends on the type and structure of the virus, revealing enveloped Phi6 phage as highly susceptible to the ILs action, while the non-enveloped phages PRD1 and MS2 proved completely resistant to ionic liquids. Furthermore, a comparison of results obtained for P100 and P001 phages demonstrated for the first time that the susceptibility of viruses to ionic liquids can be dependent on differences in the phage tail structure.

HRM-PCR in medical diagnostic / Technologia HRM-PCR w diagnostyce medycznej.

High resolution melting (HRM) is a method based on the identification of differences in the denaturation of PCR reaction products in the presence of fluorescent dyes. It allows for a detailed analysis of the genetic profiles. In addition this analysis is low-cost, single-step, closed-tube and has high sensitivity. HRM found applications in diagnostics, laboratory and clinical researches. This article is a literature review of the applications of HRM analysis in medicine.

High Resolution Melting Analysis (HRM-PCR) - method and its application / Denaturacja DNA z wysoka rozdzielczoscia (HRM-PCR) - metoda i jej zastosowanie.

DNA denaturation with High Resolution Melting PCR-HRM is a method based on the identification of differences in the denaturation of PCR reaction products in the presence of fluorescent dyes. It is used to detect genetic variation in nucleic acid sequences in many branches of science, medicine and industry. This article is a review of the current literature of the methodology, applications and development of HRM analysis, which, thanks to its advantages such as speed, low cost, flexibility and simplicity, has found many applications, and its spectrum is still expanding.

Morpholinium-based ionic liquids show antimicrobial activity against clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multi-drug resistant (MDR) pathogen. It is classified by WHO as one of the most life-threatening pathogens causing nosocomial infections. Some of its clinical isolates and their subpopulations show high persistence to many antibiotics that are recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Thus, there is a need for non-traditional classes of antibiotics to fight the increasing threat of MDR P. aeruginosa. Ionic liquids (IL) are one such promising class of novel antibiotics. We selected four strains of P. aeruginosa and studied the growth inhibition and other effects of 12 different ILs. We used the well-characterized P. aeruginosa PAO1 (ATCC 15692) as model strain and compared it to three other isolates from chronic lung infection (LES B58), skin burn infection (UCBPP-PA14) and keratitis infection (39016), respectively. The ILs consisted of either 4,4-didecylmorpholinium [Dec2Mor]+ or 4-decyl-4-ethylmorpholinium [DecEtMor]+ cations combined with different anions. We found that the ILs with 4,4-didecylmorpholinium [Dec2Mor]+ cations most effectively inhibited bacterial growth as well as reduced strain fitness and virulence factor production. Our results indicate that these ILs could be used to treat P. aeruginosa infections.

Development of high-resolution melting PCR (HRM-PCR) assay to identify native fungal species associated with the wheat endosphere.

Understanding the complexity and biodiversity of fungal communities associated with the wheat endosphere can facilitate the identification of novel strains that might be beneficial to the host plant. However, the differentiation and taxonomic classification of the endosphere-associated fungi with respect to various cultivars and plant organs are challenging, time-consuming, and expensive, even with the use of molecular techniques. In the present work, we describe a fast, simple, and low-cost method based on high-resolution melting PCR (HRM-PCR) for the identification and differentiation of wheat endogenous fungal isolates. Using this approach, we differentiated 28 fungal isolates, which belonged to five different genera, namely Alternaria, Penicillium, Epicoccum, Fusarium, and Trichoderma. Furthermore, the results of the study revealed that this method can allow large-scale screening of cultured samples.

A benzimidazole-based ruthenium(IV) complex inhibits Pseudomonas aeruginosa biofilm formation by interacting with siderophores and the cell envelope, and inducing oxidative stress.

Pseudomonas aeruginosa biofilm-associated infections are a serious medical problem, and new compounds and therapies acting through novel mechanisms are much needed. Herein, the authors report a ruthenium(IV) complex that reduces P. aeruginosa PAO1 biofilm formation by 84%, and alters biofilm morphology and the living-to-dead cell ratio at 1 mM concentration. Including the compound in the culture medium altered the pigments secreted by PAO1, and fluorescence spectra revealed a decrease in pyoverdine. Scanning electron microscopy showed that the ruthenium complex did not penetrate the bacterial cell wall, but accumulated on external cell structures. Fluorescence quenching experiments indicated strong binding of the ruthenium complex to both plasmid DNA and bovine serum albumin. Formamidopyrimidine DNA N-glycosylase (Fpg) protein digestion of plasmid DNA isolated after ruthenium(IV) complex treatment revealed the generation of oxidative stress, which was further proved by the observed upregulation of catalase and superoxide dismutase gene expression.

Adaptation of bacteria Escherichia coli in presence of quaternary ammonium ionic liquids.

This paper presents the adaptation of Escherichia coli Gram-negative bacteria to increased concentrations of ionic liquids. Theophylline-based quaternary ammonium salts were used as an example of an ionic liquid that on the one hand includes an anion of natural origin and on the other hand is characterized by amphiphilic properties due to aliphatic chains in its structure. Theophylline-based ionic liquids can be synthesized relatively cheaply and easily and can exhibit strong antibacterial properties depending on the alkyl chain length. These compounds can also strongly affect bacterial membrane properties, including changes in electrokinetic potential as well as net surface charge. The experiments performed in this study succeeded in obtaining bacterial cultures growing at a tetradecyltrimethylammonium theophyllinate concentration three times higher than the minimum inhibition and bactericidal concentration. The adapted bacteria were characterized by intriguing changes in morphology and grew in the form of almost one-millimeter spheres in a liquid medium. It was shown that cultivation of adapted bacteria with tetradecyltrimethylammonium theophyllinate resulted in changes in the lipid membrane composition and protein patterns of the bacterial lysates, depending on the ionic liquid concentration. This study also revealed that such bacterial adaptation can increase sensitivity to antibiotics by affecting membrane properties like ionophores. These results can be potentially important with regard to synergistic or antagonistic action with other bactericidal compounds like antibiotics and nanoparticles.

Bacterial Communities from the Arsenic Mine in Zloty Stok, Sudety Mountains, Poland.

Investigations of bacterial communities and characterization of mineralogy of the environment in the Zloty Stok As-Au deposit were carried out. PXRD analysis revealed the presence of picropharmacolite as the most common secondary arsenic mineral in the mine. Total DNA was extracted from slime streams or slime biofilms samples to investigate the bacterial communities. PCR amplification of 16S rDNA was performed followed by subcloning of its products. Over 170 clones were analyzed by means of RFLP method. Eight group of clones representing different restriction patterns were identified. The nucleotide sequences of their inserts suggest that bacteria present in the mine environment belong to: Flavobacteria, Sphingobacteriia, Bacteroides, Proteobacteria, Mollicutes and Firmicutes. The metagenomic approach allows to demonstrate a higher diversity of microbiota than classical microbiological studies of cultivable isolates.

Crotalarieae and Genisteae of the South African Great Escarpment are nodulated by novel Bradyrhizobium species with unique and diverse symbiotic loci.

The genus Bradyrhizobium contains predominantly nitrogen-fixing legume symbionts. Phylogenetic analysis of the genes responsible for their symbiotic abilities (i.e., those encoded on the nodulation [nod] and nitrogen-fixation [nif] loci) has facilitated the development of an extensive phylogeographic framework for the genus. This framework however contains only a few nodulating isolates from Africa. Here we focused on nodulating Bradyrhizobium isolates associated with native southern African legumes in the tribes Genisteae and Crotalarieae found along the Great Escarpment in the Mpumalanga Province of South Africa. The aims of this study were to: (1) obtain rhizobial isolates from legumes in the Genisteae and Crotalarieae; (2) verify their nodulation ability; (3) characterize them to species level based on phylogenetic analyses of several protein coding gene regions (atpD, dnaK, glnII, recA, rpoB and gyrB) and (4) determine their placement in the phylogeographic framework inferred from the sequences of the symbiotic loci nodA and nifD. Twenty of the 21 Bradyrhizobium isolates belonged to six novel species, while one was conspecific with the recently described B. arachidis. Among these isolates, the nodA phylogeny revealed several new clades, with 18 of our isolates found in Clades XIV and XV, and only three forming part of the cosmopolitan Clade III. These strains formed predominantly the same groups in the nifD phylogeny although with slight differences; indicating that both vertical and horizontal inheritance of the symbiotic loci occurred. These findings suggest that the largely unexplored diversity of indigenous African rhizobia are characterized by unique ancestries that might mirror the distribution of their hosts and the environmental factors driving their evolution.

Different antibacterial activity of novel theophylline-based ionic liquids - Growth kinetic and cytotoxicity studies.

The aim of this study was to investigate novel theophylline-based ionic liquids and their cytotoxic effects towards model Gram-positive and Gram-negative bacteria (Bacillus cereus and Escherichia coli, respectively). Growth kinetics, respiratory rates and dehydrogenase activities were studied in the presence of ionic liquids at concentrations ranging from 10 to 1000mg/L. Additionally, the influence of ionic liquids on bacterial cells associated with specific interactions based on the structure of cell wall was evaluated. This effect was assessed by viability tests and scanning electron microscope observations. The obtained results confirmed that ionic liquids exhibit different levels of toxicity in relation to Gram-positive and Gram-negative bacteria. Those effects are associated with the chemical structure of the cationic species of the ionic liquids and their critical micelle concentration value. It was established that the presence of an alkyl or allyl group increased the toxicity, whereas the presence of an aryl group in the cation decreased the toxic effect of ILs. Results presented in this study also revealed unexpected effects of self-aggregation of E. coli cells. Overall, it was established that the studied ILs exhibited higher toxicity towards Gram-positive bacteria due to different interactions between the ILs and the cell membranes. These findings may be of importance for the design of ILs with targeted antimicrobial properties.

Monitoring of Virulence Genes, Drug-Resistance in Campylobacter coli Isolated from Golden Retrievers.

The investigation was performed on 75 of Golden Retriever puppies. Faecal samples were collected on the 42 day of the puppies life (control). Probiotic preparation was administered on 43 day of the puppies life and 10 days after the application of the probiotic, faecal samples were collected again (on 53 day of puppies life). All isolates of Campylobacter coli isolated prior to the administration of the probiotic were found to contain the cadF gene responsible for adhesion, as well as, the flaA gene influencing motility of the examined bacteria. Significant differences (P < 0.05) were recorded only in the case of enrofloxacin.

Synthesis of SiC/Ag/Cellulose Nanocomposite and Its Antibacterial Activity by Reactive Oxygen Species Generation.

We describe the synthesis of nanocomposites, based on nanofibers of silicon carbide, silver nanoparticles, and cellulose. Silver nanoparticle synthesis was achieved with chemical reduction using hydrazine by adding two different surfactants to obtain a nanocomposite with silver nanoparticles of different diameters. Determination of antibacterial activity was based on respiration tests. Enzymatic analysis indicates oxidative stress, and viability testing was conducted using an epifluorescence microscope. Strong bactericidal activity of nanocomposites was found against bacteria Escherichia coli and Bacillus cereus, which were used in the study as typical Gram-negative and Gram-positive bacteria, respectively. It is assumed that reactive oxygen species generation was responsible for the observed antibacterial effect of the investigated materials. Due to the properties of silicon carbide nanofiber, the obtained nanocomposite may have potential use in technology related to water and air purification. Cellulose addition prevented silver nanoparticle release and probably enhanced bacterial adsorption onto aggregates of the nanocomposite material.

Monitoring of Virulence Genes, Drug-Resistance in Campylobacter coli Isolated from Golden Retrievers.

The investigation was performed on 75 of Golden Retriever puppies. Faecal samples were collected on the 42 day of the puppies life (con-trol). Probiotic preparation was administered on 43 day of the puppies life and 10 days after the application of the probiotic, faecal samples were collected again (on 53 day of puppies life). All isolates of Campylobacter coli isolated prior to the administration of the probiotic were found to contain the cadF gene responsible for adhesion, as well as, the flaA gene influencing motility of the examined bacteria. Significant differences (P < 0.05) were recorded only in the case of enrofloxacin.

The Prevalence of Campylobacter spp. and Occurrence of Virulence Genes Isolated from Dogs.

This study was conducted to determine the prevalence of Campylobacter spp. isolated from dogs' faecal samples. From June 2012 to June 2013, a total of 210 faecal samples from pet dogs living in different kennels (n = 210) were collected by the owners in Greater Poland Voivodeship, Poznan District, Poland. The study revealed that 105 out of 210 faecal samples (50%) contained Campylobacter. The highest prevalence of Campylobacter spp. occurred in spring (81%), followed by winter (64%). The cadF gene was found in 100% of the isolates tested. The occurrence of the other genes was variable. The isolates from young dogs were characterised by higher occurrence of virulence genes.

Oxidative stress in bacteria (Pseudomonas putida) exposed to nanostructures of silicon carbide.

Silicon carbide (SiC) nanostructures produced by combustion synthesis can cause oxidative stress in the bacterium Pseudomonas putida. The results of this study showed that SiC nanostructures damaged the cell membrane, which can lead to oxidative stress in living cells and to the loss of cell viability. As a reference, micrometric SiC was also used, which did not exhibit toxicity toward cells. Oxidative stress was studied by analyzing the activity of peroxidases, and the expression of the glucose-6-phosphate dehydrogenase gene (zwf1) using real-time PCR and northern blot techniques. Damage to nucleic acid was studied by isolating and hydrolyzing plasmids with the formamidopyrimidine [fapy]-DNA glycosylase (also known as 8-oxoguanine DNA glycosylase) (Fpg), which is able to detect damaged DNA. The level of viable microbial cells was investigated by propidium iodide and acridine orange staining.

Adsorption studies of the gram-negative bacteria onto nanostructured silicon carbide.

In this study, we demonstrated a significant adsorption of Pseudomonas putida bacteria onto aggregates of nanofibers (NFSiC) and nanorods (NRSiC) of silicon carbide (SiC) in aqueous suspensions. Langmuir and Freundlich isotherms were used to quantify adsorption affinities. It was found that adsorption of the bacteria strongly depended on the structure of the silicon carbide and the pH of the aqueous solution, which affected the isoelectric point of both the silicon carbide and the bacterial cells. The strongest affinity of bacteria was noted in the case of NRSiC aggregates. Affinity was inversely proportional to pH. Similarly, the adsorption of bacteria to the surface of the aggregates increased with decreasing pH. For NFSiC, the affinity of the bacteria for the surface of the aggregates was also inversely proportional to pH. However, adsorption increased at higher pH values. This discrepancy was explained by microscopic analysis, which showed that the bacterial cells were both adsorbed onto and trapped by NFSiC. The adsorption of bacteria onto a micrometric silicon carbide reference material was significantly smaller than adsorption onto nanostructured SiC.