Diversity and potential plant growth promoting capacity of seed endophytic bacteria of the holoparasite Cistanche phelypaea (Orobanchaceae).

Salt marshes are highly dynamic, biologically diverse ecosystems with a broad range of ecological functions. We investigated the endophytic bacterial community of surface sterilized seeds of the holoparasitic Cistanche phelypaea growing in coastal salt marshes of the Iberian Peninsula in Portugal. C. phelypaea is the only representative of the genus Cistanche that was reported in such habitat. Using high-throughput sequencing methods, 23 bacterial phyla and 263 different OTUs on genus level were found. Bacterial strains belonging to phyla Proteobacteria and Actinobacteriota were dominating. Also some newly classified or undiscovered bacterial phyla, unclassified and unexplored taxonomic groups, symbiotic Archaea groups inhabited the C. phelypaea seeds. γ-Proteobacteria was the most diverse phylogenetic group. Sixty-three bacterial strains belonging to Bacilli, Actinomycetes, α-, γ- and ß-Proteobacteria and unclassified bacteria were isolated. We also investigated the in vitro PGP traits and salt tolerance of the isolates. Among the Actinobacteria, Micromonospora spp. showed the most promising endophytes in the seeds. Taken together, the results indicated that the seeds were inhabited by halotolerant bacterial strains that may play a role in mitigating the adverse effects of salt stress on the host plant. In future research, these bacteria should be assessed as potential sources of novel and unique bioactive compounds or as novel bacterial species.

Genomes comparison of two Proteus mirabilis clones showing varied swarming ability.

BACKGROUND: Proteus mirabilis is a Gram-negative bacteria most noted for its involvement with catheter-associated urinary tract infections. It is also known for its multicellular migration over solid surfaces, referred to as 'swarming motility'. Here we analyzed the genomic sequences of two P. mirabilis isolates, designated K38 and K39, which exhibit varied swarming ability. METHODS AND RESULTS: The isolates genomes were sequenced using Illumina NextSeq sequencer, resulting in about 3.94 Mbp, with a GC content of 38.6%, genomes. Genomes were subjected for in silico comparative investigation. We revealed that, despite a difference in swarming motility, the isolates showed high genomic relatedness (up to 100% ANI similarity), suggesting that one of the isolates probably originated from the other. CONCLUSIONS: The genomic sequences will allow us to investigate the mechanism driving this intriguing phenotypic heterogeneity between closely related P. mirabilis isolates. Phenotypic heterogeneity is an adaptive strategy of bacterial cells to several environmental pressures. It is also an important factor related to their pathogenesis. Therefore, the availability of these genomic sequences will facilitate studies that focus on the host-pathogen interactions during catheter-associated urinary tract infections.

Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Isolates in Terms of Efficacy against Cereal Ground Beetle Zabrus tenebrioides (Coleoptera: Carabidae): Morphometry and Principal Component Analysis.

One of the most dangerous pests of cereals is Zabrus tenebrioides and, in Poland, it is becoming a serious pest. Entomopathogenic nematodes (EPNs) seem to be a very promising, biological control agent for this pest. Native EPN populations are well adapted to local environmental conditions. The current study characterized three Polish isolates of the EPN Steinernema feltiae, which differed in their effectiveness against Z. tenebrioides. In the field, isolate iso1Lon reduced the pest population by 37%, compared with 30% by isolate iso1Dan and 0% by the iso1Obl isolate; the number of plants damaged by Z. tenebrioides in the presence of the different isolates reflected the results in terms of the decrease in pest population size. After incubation in the soil for 60 days, recovered EPN juveniles of all three isolates were able to infect 93-100% of the test insects, with isolate iso1Obl again showing the lowest effectiveness. The juveniles of isolate iso1Obl were also morphometrically distinct from the other two isolates, as revealed by principal component analysis (PCA), which helped to distinguish the EPN isolates. These findings showed the value of using locally adapted isolates of EPNs; two of the three isolates randomly selected from Polish soil outperformed a commercial population of S. feltiae.

Bacterial Motility and Its Role in Skin and Wound Infections.

Skin and wound infections are serious medical problems, and the diversity of bacteria makes such infections difficult to treat. Bacteria possess many virulence factors, among which motility plays a key role in skin infections. This feature allows for movement over the skin surface and relocation into the wound. The aim of this paper is to review the type of bacterial movement and to indicate the underlying mechanisms than can serve as a target for developing or modifying antibacterial therapies applied in wound infection treatment. Five types of bacterial movement are distinguished: appendage-dependent (swimming, swarming, and twitching) and appendage-independent (gliding and sliding). All of them allow bacteria to relocate and aid bacteria during infection. Swimming motility allows bacteria to spread from 'persister cells' in biofilm microcolonies and colonise other tissues. Twitching motility enables bacteria to press through the tissues during infection, whereas sliding motility allows cocci (defined as non-motile) to migrate over surfaces. Bacteria during swarming display greater resistance to antimicrobials. Molecular motors generating the focal adhesion complexes in the bacterial cell leaflet generate a 'wave', which pushes bacterial cells lacking appendages, thereby enabling movement. Here, we present the five main types of bacterial motility, their molecular mechanisms, and examples of bacteria that utilise them. Bacterial migration mechanisms can be considered not only as a virulence factor but also as a target for antibacterial therapy.

Loop-Mediated Isothermal Amplification of DNA (LAMP) as an Alternative Method for Determining Bacteria in Wound Infections.

The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.

Searching for serum biomarkers linking coronary heart disease and Helicobacter pylori infection using infrared spectroscopy and artificial neural networks.

Helicobacter pylori (Hp) Gram-negative bacteria cause gastritis or gastric ulcers. They may be involved in the development of systemic diseases i.e. coronary heart disease (CHD). Both Hp infection and CHD are related to inflammation accompanied by C-reactive protein (CRP), tumor necrosis factor alfa (TNF-α) and homocysteine. Low density lipoprotein (LDL) and triglicerides are a classic risk factors of CHD. Infrared spectroscopy has been introduced for monitoring chronic infections or endogenous disorders using specific absorption bands for biocomponents typed as diagnostic markers. In this study we selected specific motives of infrared radiation (IR) spectra for the sera from CHD patients infected with Hp. In total 141 sera were used: 90 from patients with CHD, all Hp positive, and 51 from healthy donors, 32 Hp negative and 21 Hp positive. Hp status was evaluated by anti-Hp IgG antibodies and/or 13C urea breath testing. IR spectra were measured using FT-IR/FT-NIR Spectrum 400 spectrometer (PerkinElmer) chemometrically analyzed using artificial neural networks and they showed differences in absorption bands corresponding to triglicerides, CRP, homocysteine, LDL and TNF-α, and selected component groups between CHD patients infected with Hp vs healthy uninfected donors (96.15% accuracy). Triglicerides and CRP were the best biomarkers linking Hp infection with CHD.

Into the understanding the multicellular lifestyle of Proteus mirabilis on solid surfaces.

Indwelling urinary catheterization can lead to the development of catheter-associated urinary tract infections (CAUTIs), an important type of nosocomial infection, as well as other medical issues among institutionalized adults. Recently, Proteus mirabilis was highlighted as the important cause of CAUTIs. The pathogenicity of P. mirabilis is dependent on two multicellular types of surface colonization: the adherence and swarming motility. Adhesion, mostly mediated by fimbrial and nonfimbrial adhesins, is important for the initiation of biofilm formation. Moreover, the production of urease frequently results in biofilm crystallization, which leads to the blockage of catheters. The heterologous polymeric matrix of the biofilm offers protection against antibiotics and the host immune system. P. mirabilis displays remarkable motility abilities. After contact with solid surfaces, hyper-flagellated cells are able to rapidly migrate. The importance of swarming motility in CAUTIs development remains controversial; however, it was indicated that swarming cells were able to co-express other virulence factors. Furthermore, flagella are strong immunomodulating proteins. On the other hand, both biofilm formation and swarming motility implicates multiple inter- and intraspecies interactions, which might contribute to the pathogenicity.

Phosphocholine decoration of Proteus mirabilis O18 LPS induces hydrophobicity of the cell surface and electrokinetic potential, but does not alter the adhesion to solid surfaces.

Proteus mirabilis harbours a variety of O antigens, permitting evasion of the host immune response. LPS decoration with phosphocholine increases cell surface hydrophobicity and decreases electrokinetic potential, which may interfere with antibody interaction and bacterial surface recognition. The decoration does not influence adherence to solid surfaces.

Recent advances on smart glycoconjugate vaccines in infections and cancer.

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as "tumor-associated carbohydrate antigens". Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy.

Correlations between autoantibodies and the ATR-FTIR spectra of sera from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases worldwide. Due to high heterogeneity in disease manifestation, accurate and fast diagnosis of RA is difficult. This study analyzed the potential relationship between the infrared (IR) spectra obtained by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and the presence of autoantibodies and antibodies against urease in sera. Additionally, the wave number of the IR spectrum that enabled the best differentiation between patients and healthy blood donors was investigated. Using a mathematical model involving principal component analysis and discriminant analysis, it was shown that the presence of anti-citrullinated protein antibody, rheumatoid factor, anti-neutrophil cytoplasmic antibodies, and anti-nuclear antibodies correlated significantly with the wave numbers in the IR spectra of the tested sera. The most interesting findings derived from determination of the best predictors for distinguishing RA. Characteristic features included an increased reaction with urease mimicking peptides and a correspondence with particular nucleic acid bands. Taken together, the results demonstrated the potential application of ATR-FTIR in the study of RA and identified potential novel markers of the disease.

Comparison of Biological Activity of Field Isolates of Steinernema feltiae with a Commercial S. feltiae Biopesticide Product.

Insect trap studies were carried out to determine the presence of entomopathogenic nematodes (EPN) from the family Steinernematidae in the soils of Poland and to compare the biological activities of field nematode isolates with nematodes from commercial biopesticide. The fauna of these organisms in central Poland is poorly studied in both taxonomic and biological terms. Tilled soils representative of this region were sampled from cultivated fields. EPN were isolated from soil samples under laboratory conditions and identified using a key for species identification and molecular analysis. Basic morphometric parameters of infective juveniles and adult males of the first generation were determined. The research showed that males and infective juveniles Steinernema feltiae from Loniów were the largest. The smallest infective juveniles were found in the isolate from Oblasy, and the smallest males in the isolate from Danków. In Poland, new field isolates showed close genetic similarity to other S. feltiae isolates. The research showed that the field isolates from Poland had greater infectivity and rate of reproduction compared with nematodes from the commercial biopesticide. The findings indicate the potential use of field S. feltiae isolates from Poland (iso1Lon, iso1Dan and iso1Obl) to develop new biopesticide products.

Emerging glyco-based strategies to steer immune responses.

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell-cell communications and in the regulation of immune responses. Through the interaction with glycan-binding receptors, glycans are able to affect the activation status of antigen-presenting cells, leading either to induction of pro-inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco-chemists and glyco-immunologists to develop glycan-based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan-modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen-presenting cells, like dendritic cells. In addition, we address how glycan-based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan-based therapies, including chimeric antigen receptor (CAR)-T cells to achieve targeting of tumor-associated glycan-specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity.

Antibodies Isolated from Rheumatoid Arthritis Patients against Lysine-Containing Proteus mirabilis O3 (S1959) Lipopolysaccharide May React with Collagen Type I.

Most rheumatic diseases, including rheumatoid arthritis (RA), are characterized by immune disorders that affect antibody activity. In the present study, using Dot blot and ELISA assay, we showed that patients with rheumatic disease produced significantly more antibodies against lipopolysaccharide (LPS) P. mirabilis O3 compared to healthy donors (p < 0.05), and affinity purified antibodies against LPS O3 may cross-react with collagen type I. It was demonstrated that purified of antibodies isolated from RA patients sera, reacted stronger with the collagen than healthy donors (p = 0.015), and cross-reaction was correlated with level of anti-citrullinated peptide antibodies (r = 0.7, p = 0.003). Moreover, using six different lipopolysaccharides were demonstrated the significant correlations in sera reactivity among lysine-containing lipopolysaccharides observed in patients' sera (p < 0.05). Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) it was shown that unique wavenumbers of sera spectra correlate with reactivity with lipopolysaccharides allowing distinguish patients from healthy blood donors. Antibodies adsorption by synthetic antigens shows that in patients' group anti-LPS O3 antibodies can be adsorbed by both amides of galacturonic acid and lysine or threonine, which suggests less specificity of antibodies binding with non-carbohydrate LPS component. The observed correlations suggest that non-carbohydrate components of LPS may be an important epitope for less specific anti-LPS antibodies, which might lead to cross-reactions and affect disease development.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (FTIR) and Artificial Neural Networks Applied to Investigate Quantitative Changes of Selected Soluble Biomarkers, Correlated with H. pylori Infection in Children and Presumable Consequent Delayed Growth.

Helicobacter pylori infections causing gastroduodenal disorders are a common medical problem. The aim of this study was to determine the specific motives of infrared spectroscopy (IR) spectra of sera from H. pylori-infected and uninfected children applied to investigate quantitatively-selected soluble biomarkers correlated with H. pylori infection in children and presumable consequent delayed growth. Sera from 41 children infected with H. pylori (Hp(+)) and 43 uninfected (Hp(-)) under the care of the Polish Mother's Hospital in Lodz, Poland, were analyzed. The H. pylori status was confirmed by gastroscopy, 13C urea breath testing, and anti-H. pylori IgG antibodies. Infrared spectra were measured using an FTIR/FT-NIR Spectrum 400 spectrometer (PerkinElmer). The IR spectrum was measured in the wavenumber range 3000-750 cm-1 and subjected to mathematical calculation of the first derivative. Based on the chi-square test, 10 wavenumbers of spectra correlating with H. pylori infection were selected for use in designing an artificial neural network. Ten parts of the IR spectra correlating with H. pylori infection were identified in the W2 and W3 windows associated mainly with proteins and the W4 window related to nucleic acids and hydrocarbons. Artificial neural networks for H. pylori infection were developed based on chemometric data. By mathematical modeling, children were classified towards H. pylori infection in conjunction with elevated levels of selected biomarkers in serum potentially related to growth retardation. The study concludes that IR spectroscopy and artificial neural networks may help to confirm H. pylori-driven growth disorders in children.

Use of Fourier-Transform Infrared Spectroscopy (FT-IR) for Monitoring Experimental Helicobacter pylori Infection and Related Inflammatory Response in Guinea Pig Model.

Infections due to Gram-negative bacteria Helicobacter pylori may result in humans having gastritis, gastric or duodenal ulcer, and even gastric cancer. Investigation of quantitative changes of soluble biomarkers, correlating with H. pylori infection, is a promising tool for monitoring the course of infection and inflammatory response. The aim of this study was to determine, using an experimental model of H. pylori infection in guinea pigs, the specific characteristics of infrared spectra (IR) of sera from H. pylori infected (40) vs. uninfected (20) guinea pigs. The H. pylori status was confirmed by histological, molecular, and serological examination. The IR spectra were measured using a Fourier-transform (FT)-IR spectrometer Spectrum 400 (PerkinElmer) within the range of wavenumbers 3000-750 cm-1 and converted to first derivative spectra. Ten wavenumbers correlated with H. pylori infection, based on the chi-square test, were selected for a K-nearest neighbors (k-NN) algorithm. The wavenumbers correlating with infection were identified in the W2 and W3 windows associated mainly with proteins and in the W4 window related to nucleic acids and hydrocarbons. The k-NN for detection of H. pylori infection has been developed based on chemometric data. Using this model, animals were classified as infected with H. pylori with 100% specificity and 97% sensitivity. To summarize, the IR spectroscopy and k-NN algorithm are useful for monitoring experimental H. pylori infection and related inflammatory response in guinea pig model and may be considered for application in humans.

Characterization of Proteus mirabilis Lipopolysaccharide Samples by Infrared Spectroscopy and Serological Methods.

Methods of lipopolysaccharide extraction, purification, and sample validation are presented. Based on serological reaction in ELISA, immunoblotting, and infrared spectra, identities of two LPS preparations from smooth P. mirabilis (O18) PrK 34/57 are presented.

Assessment of Proteus mirabilis Antigen Immunological Complexes by Atomic Force Microscopy.

Atomic force microscopy (AFM) is being increasingly used to directly measure protein interactions in nearly physiological environments. Here, protocols for atomic force microscopy (AFM) for visualization of antigen-antibody complexes are presented. The technique is used to demonstrate complexes formed by rheumatoid arthritis patient antibodies with lipopolysaccharide (LPS) isolated from P. mirabilis (O3) strain S1959 and a synthetic antigen (LPS epitope of 6 N-alpha-(D-galacturonoyl)-L-lysine residues).

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.

Draft Genome Sequences of Proteus mirabilis K1609 and K670: A Model Strains for Territoriality Examination.

Proteus mirabilis is a pathogenic Gram-negative bacterium characterized by its ability to swarm across surfaces, which frequently leads to colonization of the urinary tract and causes severe infections. P. mirabilis strains are also well known from their self-recognition phenomenon, referred to as Dienes phenomenon. In this study, we present novel aspect of self-recognition, which is a hierarchy in terms of strains territoriality. We report the draft genome sequences of P. mirabilis K1609 and K670 strains exhibiting the strongest and the weakest territoriality, respectively. Our results indicated that K1609 is closely related to strain BB2000, a model system for self-recognition, comparing with the K670. We annotated genes associated with recognition of kin and swarming initiation control and indicated polymorphisms by which observed differences in territoriality might results from. The phenotypic and genomic features of both strains reveal their application as a model organisms for studying not only the mechanisms of kin-recognition but also strains territoriality, thus providing new approach to the phenomenon. Availability of these genome sequences may facilitate understanding of the interactions between P. mirabilis strains.