An alternative method for SARS-CoV-2 detection with use modified fluorescent in situ hybridization.

The real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) tests are the gold standard in detecting SARS-CoV-2 virus infection. However, despite high sensitivity and specificity, they have limitations that in some cases may result in false negative results. Therefore, it is reasonable to search for additional tools that could support microbiological diagnosis of SARS-CoV-2. The aim of the study was to develop a highly specific molecular test capable of detecting and visualizing SARS-CoV-2 infection. A universal probe and a set of 18 specific oligonucleotides with a FLAP sequence attached to them on both sides were designed to visualize SARS-CoV-2 virus infection based on the fluorescence in situ hybridization method (FISH). FISH conditions using the developed kit were standardized on the Vero CCL-81 cell line infected by SARS-CoV-2 virus. The method was tested on 290 nasopharyngeal swabs (collected in a doublet) from patients with clinical symptoms of SARS-CoV-2. Each one swab from the doublet was subjected to RNA isolation and amplification by rRT-PCR. From the second swab, a microscopic preparation was performed for FISH. The use of the rRT-PCR allowed obtaining 200 positive and 90 negative results, while our FISH method allowed for 220 positive results and 70 negative results. The differences obtained using both methods were statistically significant (p = 0.008). The obtained results support the use of FISH as an additional method in microbiological diagnostics of SARS-CoV-2.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Voluntary counselling and testing points (VCTs) in Poland as a complementary and accessible alternative to public healthcare facilities: A comparative analysis of epidemiological data from the VCTs and the National Institute of Public Health NIH-National Research Institute (2015-2022).

OBJECTIVE: Voluntary counselling and testing points (VCTs) offer anonymous and free HIV tests in Poland. They also play an essential role in educational initiatives focused on the prevention and diagnosis of HIV and other sexually transmitted infections. However, no comprehensive data is available that summarizes the results of the work carried out by these VCTs. Therefore, our aim was to conduct a comparative analysis of epidemiological data obtained from VCTs and data reported by the epidemiological surveillance undertaken by the National Institute of Public Health NIH-National Research Institute (NIPH NIH-NRI) covering the period from 2015 to 2022. METHODS: This retrospective analysis was conducted on data from 258 071 people attending VCTs in Poland in 2015-2022. RESULTS: On average, 32 259 individuals underwent testing each year, with a notable increase in the number of people being tested in November. The average positivity rate was 1.39% (3576/258 071). The Masovian voivodeship conducted the most tests and had the highest number of positive results. The comparative analysis of the frequency of detecting positive results in VCTs and those reported in NIPH NIH-NRI data revealed that, on average, 31.49% (3576/11 356) of positive results in Poland between 2015 and 2022 were identified through tests conducted at VCTs. CONCLUSION: The positive results identified in VCTs constituted approximately one-third of all results reported by the National Institute of Public Health NIH-National Research Institute, highlighting the importance of VCTs. Moreover, the high availability of testing in the Masovian voivodeship resulted in better detection of HIV. The educational actions performed during European Testing Week increased the number of tests performed in November.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Improving Biocompatibility of Polyurethanes Apply in Medicine Using Oxygen Plasma and Its Negative Effect on Increased Bacterial Adhesion.

Polyurethanes (PUs) are versatile polymers used in medical applications due to their high flexibility and fatigue resistance. PUs are widely used for synthetic blood vessels, wound dressings, cannulas, and urinary and cardiovascular catheters. Many scientific reports indicate that surface wettability is crucial for biocompatibility and bacterial adhesion. The use of oxygen plasma to modify PUs is advantageous because of its effectiveness in introducing oxygen-containing functional groups, thereby altering surface wettability. The purpose of this study was to investigate the effect of the modification of the oxygen plasma of polyurethane on its biocompatibility with lung tissue (A549 cell line) and the adhesion of Gram-positive bacteria (S. aureus and S. epidermidis). The results showed that the modification of polyurethane by oxygen plasma allowed the introduction of functional groups containing oxygen (-OH and -COOH), which significantly increased its hydrophilicity (change from 105° ± 2° to 9° ± 2°) of PUs. Surface analysis by atomic force microscopy (AFM) showed changes in PU topography (change in maximum height from ∼110.3 nm to ∼32.1 nm). Moreover, biocompatibility studies on A549 cells showed that on the PU-modified surface, the cells exhibited altered morphology (increases in cell surface area and length, and thus reduced circularity) without concomitant effects on cell viability. However, serial dilution and plate count and microscopic methods confirmed that plasma modification significantly increased the adhesion of S. aureus and S. epidermidis bacteria. This study indicate the important role of surface hydrophilicity in biocompatibility and bacterial adhesion, which is important in the design of new medical biomaterials.

Standardization of the protocol for oral cavity examination and collecting of the biological samples for microbiome research using the next-generation sequencing (NGS): own experience with the COVID-19 patients.

To date, publications have shown that compositions of oral microbiota differ depending on their habitats (e.g. tongue, tonsils, pharynx). The absence of set standards for the choice of the areas and conditions of material collection makes the oral microbiome one of the most difficult environments for a comparative analysis with other researchers, which is a meaningful limitation during an assessment of the potential effects of microorganisms as biomarkers in the courses of various human diseases. Therefore, standardisation of basic conditions of a dental examination and collection of material for the next generation sequencing (NGS) is worth attempting. The standardisation of the dental exam and collection of the clinical materials: saliva, swab from the tongue ridge, hard palate, palatine tonsils and oropharynx, supragingival plaque and subgingival plaque. Protocol involved the patients (n = 60), assigned to 3 groups: I-COVID-19 convalescents who received antibiotics, n = 17, II-COVID-19 convalescents, n = 23 and III-healthy individuals, n = 20. The collected biological samples were used to conduct NGS (16S rRNA). The conditions of patient preparation for collecting biological materials as well as the schedule of dental examination, were proposed. Based on the research conducted, we have indicated the dental indicators that best differentiate the group of COVID-19 patients (groups I and II) from healthy people (group III). These include the DMFT, D and BOP indices. The use of alpha and beta diversity analysis provided an overall insight into the diversity of microbial communities between specific niches and patient groups. The most different diversity between the studied group of patients (group II) and healthy people (group III) was noted in relation to the supragingival plaque. The order of activities during the dental exam as well as while collecting and securing clinical materials is particularly important to avoid technical errors and material contamination which may result in erroneous conclusions from the analyses of the results of sensitive tests such as the NGS. It has been shown that the dental indices: DMFT, D number, PI and BOP are the best prognostic parameters to assess the oral health. Based on beta diversity the most sensitive niche and susceptible to changes in the composition of the microbiota is the supragingival plaque. The procedures developed by our team can be applied as ready-to-use forms in studies conducted by other researchers.

Revealing bactericidal events on graphene oxide nano films deposited on metal implant surfaces.

At the time when pathogens are developing robust resistance to antibiotics, the demand for implant surfaces with microbe-killing capabilities has significantly risen. To achieve this goal, profound understanding of the underlying mechanisms is crucial. Our study demonstrates that graphene oxide (GO) nano films deposited on stainless steel (SS316L) exhibit superior antibacterial features. The physicochemical properties of GO itself play a pivotal role in influencing biological events and their diversity may account for the contradictory results reported elsewhere. However, essential properties of GO coatings, such as oxygen content and the resulting electrical conductivity, have been overlooked so far. We hypothesize that the surface potential and electrical resistance of the oxygen content in the GO-nano films may induce bacteria-killing events on conductive metallic substrates. In our study, the GO applied contains 52 wt% of oxygen, and thus exhibits insulating properties. When deposited as a nano film on an electrically conducting steel substrate, GO flakes generate a Schottky barrier at the interface. This barrier, consequently, impedes the transfer of electrons to the underlying conductive substrate. As a result, this creates reactive oxygen species (ROS), leading to bacterial death. We confirmed the presence of GO coatings and their hydrolytic stability by using X-ray photoelectron spectroscopy (XPS), µRaman spectroscopy, scanning electron microscopy (SEM), and Kelvin probe force microscopy (KPFM) measurements. The biological evaluation was performed on the MG63 osteoblast-like cell line and two selected bacteria species: S. aureus and P. aeruginosa, demonstrating both the cytocompatibility and antibacterial behavior of GO-coated SS316L substrates. We propose a two-step bactericidal mechanism: electron transfer from the bacteria membrane to the substrate, followed by ROS generation. This mechanism finds support in changes observed in contact angle, surface potential, and work function, identified as decisive factors. By addressing overlooked factors and effectively bridging the gap between understanding and practicality, we present a transformative approach for implant surfaces, combating microbial resistance, and offering new application possibilitie.

In Vitro and In Silico Studies of Functionalized Polyurethane Surfaces toward Understanding Biologically Relevant Interactions.

The solid-aqueous boundary formed upon biomaterial implantation provides a playground for most biochemical reactions and physiological processes involved in implant-host interactions. Therefore, for biomaterial development, optimization, and application, it is essential to understand the biomaterial-water interface in depth. In this study, oxygen plasma-functionalized polyurethane surfaces that can be successfully utilized in contact with the tissue of the respiratory system were prepared and investigated. Through experiments, the influence of plasma treatment on the physicochemical properties of polyurethane was investigated by atomic force microscopy, attenuated total reflection infrared spectroscopy, differential thermal analysis, X-ray photoelectron spectroscopy, secondary ion mass spectrometry, and contact angle measurements, supplemented with biological tests using the A549 cell line and two bacteria strains (Staphylococcus aureus and Pseudomonas aeruginosa). The molecular interpretation of the experimental findings was achieved by molecular dynamics simulations employing newly developed, fully atomistic models of unmodified and plasma-functionalized polyurethane materials to characterize the polyurethane-water interfaces at the nanoscale in detail. The experimentally obtained polar and dispersive surface free energies were consistent with the calculated free energies, verifying the adequacy of the developed models. A 20% substitution of the polymeric chain termini by their oxidized variants was observed in the experimentally obtained plasma-modified polyurethane surface, indicating the surface saturation with oxygen-containing functional groups.

Oral microbiota study of the patients after hospitalisation for COVID-19, considering selected dental indices and antibiotic therapy using the next generation sequencing method (NGS).

Background: Poor oral hygiene and the increased incidence and severity of periodontitis may exacerbate SARS-CoV-2 infection. The aim was to evaluate the oral microbiota of 60 participants divided into groups: COVID-19 convalescents who received antibiotics during hospitalization (I), COVID-19 convalescents without antibiotic therapy (II) and healthy individuals (III). Materials and Methods: Dental examination was conducted, and oral health status was evaluated using selected dental indexes. Clinical samples (saliva, dorsal swabs, supragingival and subgingival plaque) were collected and used for metagenomic library to the next-generation sequencing (NGS) preparation. Results: Each of the clinical materials in particular groups of patients showed a statistically significant and quantitatively different bacterial composition. Patients from group I showed significantly worse oral health, reflected by higher average values of dental indexes and also a higher percentage of Veillonella, Tannerella, Capnocytophaga and Selenomonas genera in comparison to other groups. Additionally, a statistically significant decrease in the amount of Akkermansia type in both groups with COVID-19 was observed for all materials. Conclusions: The primary factor affecting the composition of oral microbiota was not the SARS-CoV-2 infection itself, but the use of antibiotic therapy. The increased percentage of pro-inflammatory pathogens observed in COVID-19 patients underscores the importance of preventing periodontal disease and improving oral hygiene in the future.

Poly(sebacic acid) microparticles loaded with azithromycin as potential pulmonary drug delivery system: Physicochemical properties, antibacterial behavior, and cytocompatibility studies.

Recurrent bacterial infections are a common cause of death for patients with cystic fibrosis and chronic obstructive pulmonary disease. Herein, we present the development of the degradable poly(sebacic acid) (PSA) microparticles loaded with different concentrations of azithromycin (AZ) as a potential powder formulation to deliver AZ locally to the lungs. We characterized microparticle size, morphology, zeta potential, encapsulation efficiency, interaction PSA with AZ and degradation profile in phosphate buffered saline (PBS). The antibacterial properties were evaluated using the Kirby-Bauer method against Staphylococcus aureus. Potential cytotoxicity was evaluated in BEAS-2B and A549 lung epithelial cells by the resazurin reduction assay and live/dead staining. The results show that microparticles are spherical and their size, being in the range of 1-5 µm, should be optimal for pulmonary delivery. The AZ encapsulation efficiency is nearly 100 % for all types of microparticles. The microparticles degradation rate is relatively fast - after 24 h their mass decreased by around 50 %. The antibacterial test showed that released AZ was able to successfully inhibit bacteria growth. The cytotoxicity test showed that the safe concentration of both unloaded and AZ-loaded microparticles was equal to 50 µg/ml. Thus, appropriate physicochemical properties, controlled degradation and drug release, cytocompatibility, and antibacterial behavior showed that our microparticles may be promising for the local treatment of lung infections.

Sonochemical Deposition of Gentamicin Nanoparticles at the PCV Tracheostomy Tube Surface Limiting Bacterial Biofilm Formation.

BACKGROUND: The use of nanotechnology in the production of medical equipment has opened new possibilities to fight bacterial biofilm developing on their surfaces, which can cause infectious complications. In this study, we decided to use gentamicin nanoparticles. An ultrasonic technique was used for their synthesis and immediate deposition onto the surface of tracheostomy tubes, and their effect on bacterial biofilm formation was evaluated. METHODS: Polyvinyl chloride was functionalized using oxygen plasma followed by sonochemical formation and the embedment of gentamicin nanoparticles. The resulting surfaces were characterized with the use of AFM, WCA, NTA, FTIR and evaluated for cytotoxicity with the use of A549 cell line and for bacterial adhesion using reference strains of S. aureus (ATCC® 25923™) and E. coli (ATCC® 25922™). RESULTS: The use of gentamicin nanoparticles significantly reduced the adhesion of bacterial colonies on the surface of the tracheostomy tube for S. aureus from 6 × 105 CFU/mL to 5 × 103 CFU/mL and for E. coli from 1.655 × 105 CFU/mL to 2 × 101 CFU/mL, and the functionalized surfaces did not show a cytotoxic effect on A549 cells (ATTC CCL 185). CONCLUSIONS: The use of gentamicin nanoparticles on the polyvinyl chloride surface may be an additional supporting method for patients after tracheostomy in order to prevent the colonization of the biomaterial by potentially pathogenic microorganisms.

Porous Zirconia Scaffolds Functionalized with Calcium Phosphate Layers and PLGA Nanoparticles Loaded with Hydrophobic Gentamicin.

Implant-related infections are a worldwide issue that is considered very challenging. Conventional therapies commonly end up failing; thus, new solutions are being investigated to overcome this problem. The in situ delivery of the drug at the implant site appears to be more sufficient compared to systemic antibiotic therapy. In this study, we manufactured porous zirconia scaffolds using the foam replication method. To improve their overall bioactivity, they were coated with a calcium phosphate (CaP) layer containing antibiotic-loaded degradable polymer nanoparticles (NPs) obtained by the double emulsion method to achieve the antibacterial effect additionally. Encapsulation efficiency (EE) and drug loading (DL) were superior and were equal to 99.9 ± 0.1% and 9.1 ± 0.1%, respectively. Scaffolds were analyzed with scanning electron microscopy, and their porosity was evaluated. The porosity of investigated samples was over 90% and resembled the microstructure of spongy bone. Furthermore, we investigated the cytocompatibility with osteoblast-like MG-63 cells and antimicrobial properties with Staphylococcus aureus. Scaffolds coated with a CaP layer were found non-toxic for MG-63 cells. Moreover, the presence of antibiotic-loaded nanoparticles had no significant influence on cell viability, and the obtained scaffolds inhibited bacteria growth. Provided processes of fabrication of highly porous zirconia scaffolds and surface functionalization allow minimizing the risk of implant-related infection.

The Two-Track Investigation of Fibronectin Binding Protein A of Staphylococcus aureus from Bovine Mastitis as a Potential Candidate for Immunodiagnosis: A Pilot Study.

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Do NAAT-Based Methods Increase the Diagnostic Sensitivity of Streptococcus agalactiae Carriage Detection in Pregnant Women?

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33-63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.

Stethoscopes or Maybe "Bacterioscopes" - Is hand Hygiene Solely Capable of Preventing Hospital-Associated Infections?

The stethoscope remains an indispensable diagnostic tool for medical students. Improper stethoscope hygiene may cause bacterial infections, including hospital-associated infections (HAIs), which challenge the Polish medical system. The study's main objective was to evaluate the hygiene habits declared by medical students. Moreover, microbiological control with the characteristics of potentially pathogenic microorganisms was performed. The study included 66 medical students from the Faculty of Medicine at the Jagiellonian University Medical College in Cracow, Poland. The participants filled in an anonymous questionnaire. Stethoscope contamination was assessed through isolation, identification, testing of antibiotic resistance, and clonality of the isolates bacterial pathogens. The survey showed that only 30.3% of students cleaned their stethoscopes after each patient, and 1.5% never did this. Of the 66 stethoscopes tested, 100% were positive for bacterial growth. Staphylococcus spp. was the most frequently isolated contaminant (50.5%). The questionnaire results demonstrated the necessity of the validated procedures for cleaning the stethoscopes. Stethoscopes used by medical students are contaminated with numerous bacterial species, including multidrug-resistant organisms. The clonal structure of the MRSA and MRSE populations acquired from stethoscopes has been demonstrated. Our results confirm the possibility that these medical devices mediate the spread of hazardous pathogens in the hospital environment. Practical exercises are essential to forming the correct hygiene habits involving stethoscopes, which enable practicing and checking the correctness of the established skills.

New insights into diversity of the upper respiratory tract microbiota and its relationship with SARS-CoV-2 viral load in the nasopharyngeal epithelial cells in patients with COVID-19.

INTRODUCTION: The effects of SARS­CoV­2 infection on the composition of the upper respiratory tract (URT) microbiota are yet to be established, and more attention to this topic is needed. OBJECTIVES: The study aimed to assess the bacterial profile and the possible association between the URT microbiota composition and the SARS­CoV­2 viral load. PATIENTS AND METHODS: Nasopharyngeal swabs were taken from 60 adult patients with SARS­CoV­2 infection who were divided into 3 groups based on the quantification cycle (Cq) value in the quantitative polymerase chain reaction test: group I (n = 20), Cq lower than or equal to 31 (high replication rate); group II (n = 20), Cq greater than 31 and lower than 38 (low replication rate), and group III (n = 20), Cq higher than or equal to 38 (virus eliminated from the nasopharyngeal epithelial cells). The obtained genetic libraries of 16S rRNA were sequenced and taxonomic diversity profiling was performed to determine the α- and ß­biodiversity in each group. RESULTS: A significantly lower abundance of Prevotella species was noted in group I, as compared with groups II and III. Akkermansia muciniphila, Faecalibacterium prausnitzii, Fusicatenibacterium saccharivorans, and Bacteroides dorei abundance was characteristic of and significantly greater in group I than in groups II and III. Overall, the microbiota composition was the most diverse in group I, whereas groups II and III were more homogenous in terms of taxonomic diversity. CONCLUSIONS: The arbitrary division of patients according to the SARS­CoV­2 viral load was reflected in diverse composition of their bacterial microbiota, which implies an association between these 2 factors. The patients with a low viral replication rate and those who eliminated the virus from the epithelial cells belonged to a group with a less diverse microbiota community than the patients with a high viral replication rate.

Detection of immunoreactive proteins of Escherichia coli, Streptococcus uberis, and Streptococcus agalactiae isolated from cows with diagnosed mastitis.

Introduction: Mastitis is a widespread mammary gland disease of dairy cows that causes severe economic losses to dairy farms. Mastitis can be caused by bacteria, fungi, and algae. The most common species isolated from infected milk are, among others, Streptococcus spp., and Escherichia coli. The aim of our study was protein detection based on both in silico and in vitro methods, which allowed the identification of immunoreactive proteins representative of the following species: Streptococcus uberis, Streptococcus agalactiae, and Escherichia coli. Methods: The study group included 22 milk samples and 13 serum samples obtained from cows with diagnosed mastitis, whereas the control group constituted 12 milk samples and 12 serum samples isolated from healthy animals. Detection of immunoreactive proteins was done by immunoblotting, while amino acid sequences from investigated proteins were determined by MALDI-TOF. Then, bioinformatic analyses were performed on detected species specific proteins in order to investigate their immunoreactivity. Results: As a result, we identified 13 proteins: 3 (molybdenum cofactor biosynthesis protein B, aldehyde reductase YahK, outer membrane protein A) for E. coli, 4 (elongation factor Tu, tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG, GTPase Obg, glyceraldehyde-3-phosphate dehydrogenase) for S. uberis, and 6 (aspartate carbamoyltransferase, elongation factor Tu, 60 kDa chaperonin, elongation factor G, galactose-6-phosphate isomerase subunit LacA, adenosine deaminase) for S. agalactiae, which demonstrated immunoreactivity to antibodies present in serum from cows with diagnosed mastitis. Discussion: Due to the confirmed immunoreactivity, specificity and localization in the bacterial cell, these proteins can be considered considered potential targets in innovative rapid immunodiagnostic assays for bovine mastitis, however due to the limited number of examined samples, further examination is needed.

Identifying Bacteria Species on Microscopic Polyculture Images Using Deep Learning.

Preliminary microbiological diagnosis usually relies on microscopic examination and, due to the routine culture and bacteriological examination, lasts up to 11 days. Hence, many deep learning methods based on microscopic images were recently introduced to replace the time-consuming bacteriological examination. They shorten the diagnosis by 1-2 days but still require iterative culture to obtain monoculture samples. In this work, we present a feasibility study for further shortening the diagnosis time by analyzing polyculture images. It is possible with multi-MIL, a novel multi-label classification method based on multiple instance learning. To evaluate our approach, we introduce a dataset containing microscopic images for all combinations of four considered bacteria species. We obtain ROC AUC above 0.9, proving the feasibility of the method and opening the path for future experiments with a larger number of species.

Insight in Superiority of the Hydrophobized Gentamycin in Terms of Antibiotics Delivery to Bone Tissue.

Bone infections are a serious problem to cure, as systemic administration of antibiotics is not very effective due to poor bone vascularization. Therefore, many drug delivery systems are investigated to solve this problem. One of the potential solutions is the delivery of antibiotics from poly(L-actide-co-glycolide) (PLGA) nanoparticles suspended in the gellan gum injectable hydrogel. However, the loading capacity and release kinetics of the system based on hydrophilic drugs (e.g., gentamycin) and hydrophobic polymers (e.g., PLGA) may not always be satisfying. To solve this problem, we decided to use hydrophobized gentamycin obtained by ion-pairing with dioctyl sulfosuccinate sodium salt (AOT). Herein, we present a comparison of the PLGA nanoparticles loaded with hydrophobic or hydrophilic gentamycin and suspended in the hydrogel in terms of physicochemical properties, drug loading capacity, release profiles, cytocompatibility, and antibacterial properties. The results showed that hydrophobic gentamycin may be combined in different formulations with the hydrophilic one and is superior in terms of encapsulation efficiency, drug loading, release, and antibacterial efficacy with no negative effect on the NPs morphology or hydrogel features. However, the cytocompatibility of hydrophobic gentamycin might be lower, consequently more extensive study on its biological properties should be provided to evaluate a safe dose.

Recent Advances in the Control of Clinically Important Biofilms.

Biofilms are complex structures formed by bacteria, fungi, or even viruses on biotic and abiotic surfaces, and they can be found in almost any part of the human body. The prevalence of biofilm-associated diseases has increased in recent years, mainly because of the frequent use of indwelling medical devices that create opportunities for clinically important bacteria and fungi to form biofilms either on the device or on the neighboring tissues. As a result of their resistance to antibiotics and host immunity factors, biofilms have been associated with the development or persistence of several clinically important diseases. The inability to completely eradicate biofilms drastically increases the burden of disease on both the patient and the healthcare system. Therefore, it is crucial to develop innovative ways to tackle the growth and development of biofilms. This review focuses on dental- and implant-associated biofilm infections, their prevalence in humans, and potential therapeutic intervention strategies, including the recent advances in pharmacology and biomedical engineering. It lists current strategies used to control the formation of clinically important biofilms, including novel antibiotics and their carriers, antiseptics and disinfectants, small molecule anti-biofilm agents, surface treatment strategies, and nanostructure functionalization, as well as multifunctional coatings particularly suitable for providing antibacterial effects to the surface of implants, to treat either dental- or implant-related bacterial infections.