Lipidomics Characterization of the Microbiome in People with Diabetic Foot Infection Using MALDI-TOF MS.

Lipidomic profiling has emerged as a powerful tool for the comprehensive characterization of bacterial species, particularly in the context of clinical diagnostics. Utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), this study aims to elucidate the lipidomic landscapes of bacterial strains isolated from diabetic foot infections (DFI). Our analysis successfully identified a diverse array of lipids in the cellular membranes of both Gram-positive and Gram-negative bacteria, revealing a total of 108 unique fatty acid combinations. Specifically, we identified 26 LPG, 33 LPE, 43 PE, 114 PG, 89 TAG, and 120 CLP in Gram-positive bacteria and 10 LPG, 14 LPE, 124 PE, 37 PG, 13 TAG, and 22 CLP in Gram-negative strains. Key fatty acids, such as palmitic acid, palmitoleic acid, stearic acid, and oleic acid, were prominently featured. Univariate analysis further highlighted distinct lipidomic signatures among the bacterial strains, revealing elevated levels of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in Gram-negative bacteria associated with DFI. In contrast, Gram-positive strains demonstrated increased or uniquely fluctuating levels of triglyceride (TAG) and cardiolipin (CLP). These findings not only underscore the utility of MALDI-TOF MS in bacterial lipidomics but also provide valuable insights into the lipidomic adaptations of bacteria in diabetic foot infections, thereby laying the groundwork for future studies aimed at constructing microbial lipid libraries for enhanced bacterial identification.

Multi-Instrumental Analysis Toward Exploring the Diabetic Foot Infection Microbiota.

The polymicrobial nature of diabetic foot infection (DFI) makes accurate identification of the DFI microbiota, including rapid detection of drug resistance, challenging. Therefore, the main objective of this study was to apply matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) technique accompanied by multiply culture conditions to determine the microbial patterns of DFIs, as well as to assess the occurrence of drug resistance among Gram-negative bacterial isolates considered a significant cause of the multidrug resistance spread. Furthermore, the results were compared with those obtained using molecular techniques (16S rDNA sequencing, multiplex PCR targeting drug resistance genes) and conventional antibiotic resistance detection methods (Etest strips). The applied MALDI-based method revealed that, by far, most of the infections were polymicrobial (97%) and involved many Gram-positive and -negative bacterial species-19 genera and 16 families in total, mostly Enterobacteriaceae (24.3%), Staphylococcaceae (20.7%), and Enterococcaceae (19.8%). MALDI drug-resistance assay was characterized by higher rate of extended-spectrum beta-lactamases (ESBLs) and carbapenemases producers compared to the reference methods (respectively 31% and 10% compared to 21% and 2%) and revealed that both the incidence of drug resistance and the species composition of DFI were dependent on the antibiotic therapy used. MALDI approach included antibiotic resistance assay and multiply culture conditions provides microbial identification at the level of DNA sequencing, allow isolation of both common (eg. Enterococcus faecalis) and rare (such as Myroides odoratimimus) bacterial species, and is effective in detecting antibiotic-resistance, especially those of particular interest-ESBLs and carbapenemases.

Silver Nanoparticle Targets Fabricated Using Chemical Vapor Deposition Method for Differentiation of Bacteria Based on Lipidomic Profiles in Laser Desorption/Ionization Mass Spectrometry.

The global threat of numerous infectious diseases creates a great need to develop new diagnostic methods to facilitate the appropriate prescription of antimicrobial therapy. More recently, the possibility of using bacterial lipidome analysis via laser desorption/ionization mass spectrometry (LDI-MS) as useful diagnostic tool for microbial identification and rapid drug susceptibility has received particular attention because lipids are present in large quantities and can be easily extracted similar to ribosomal proteins. Therefore, the main goal of the study was to evaluate the efficacy of two different LDI techniques-matrix-assisted (MALDI) and surface-assisted (SALDI) approaches-in the classification of the closely related Escherichia coli strains under cefotaxime addition. Bacterial lipids profiles obtained by using the MALDI technique with different matrices as well as silver nanoparticle (AgNP) targets fabricated using the chemical vapor deposition method (CVD) of different AgNP sizes were analyzed by the means of different multivariate statistical methods such as principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), sparse partial least squares discriminant analysis (sPLS-DA), and orthogonal projections to latent structures discriminant analysis (OPLS-DA). The analysis showed that the MALDI classification of strains was hampered by interference from matrix-derived ions. In contrast, the lipid profiles generated by the SALDI technique had lower background noise and more signals associated with the sample, allowing E. coli to be successfully classified into cefotaxime-resistant and cefotaxime-sensitive strains, regardless of the size of the AgNPs. AgNP substrates obtained using the CVD method were used for the first time for distinguishing closely related bacterial strains based on their lipidomic profiles and demonstrate high potential as a future diagnostic tool for the detection of antibiotic susceptibility.

Viability study of clinical bacterial strains by capillary electrophoresis and flow cytometry approaches.

One of the challenges medicine faces is the constantly growing resistance of pathogens to various classes of antibiotics. In this study, we investigated the use of capillary electrophoresis (CE) to characterize and assess the physiological states of three clinical bacterial strains-methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), and Escherichia coli extended-spectrum ß-lactamases (ESßL)-exposed to different antibiotics. All chosen bacteria are the leading causes of healthcare-associated and hospital-acquired invasive infections in adults. In the first part of the research, it was determined the optimal incubation time of the tested strains with antibiotics, represented as an optimal time of 24 h. In the second part, we have compared two approaches: flow cytometry (FC) as a standard method and CE as a proposed alternative approach. The viability of clinical strains treated with different class antibiotics calculated in CE measurements was strongly correlated (>0.83 for MSSA, >0.92 for ESßL and MRSA) with the viability obtained on the basis of FC measurements. As a result, CE has a chance to become a modern diagnostic method used in clinical practice. The CE cutoff was found to be 50%; above this value, the strain shows resistance to the action of the antibiotic.

A New Approach to Imaging and Rapid Microbiome Identification for Prostate Cancer Patients Undergoing Radiotherapy.

(1) Background: Little is known about the impact of urinary microflora, in particular, its effects on side effects after radiotherapy. The use of mass spectrometry identification method (MALDI) may bring a new look at the issue of the composition and significance of the urinary microbiome. This study aimed to use the mass spectrometry identification method (MALDI) to identify the microbiome of urine samples collected from 50 irradiated prostate cancer patients. (2) Methods: Blood and urine samples were collected before gold marker implantation, at the start and last day of radiotherapy, 1, 4 months after. Patients do not always collect the urine from the midstream; therefore, samples were collected from the first void and midstream in 12 patients for MALDI analysis; in the remaining 38 patients-from the midstream void for MALDI and biochemical analysis. (3) Results: Microorganisms were present in 140/181 urine samples. We found 33 different species 3G(-) and 30G(+). The most frequently isolated strains were: Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis, Enterococcus faecalis, and Micrococcus luteus. When comparing the type of urine samples, bacteria were more common in samples from the first-void urine than from the midstream one. The absence of bacteria was found in 12.2% of samples from the first-void urine and in 24.7% from the midstream. There was no difference in the total incidence of species between streams (p = 0.85). Before fiducial implantation, the total number of detected bacterial species was significantly higher in comparison to the end of radiotherapy (p = 0.038), indicating that the administered therapy resulted in depleting the local microbiome. One month after radiotherapy, an increase in the number of isolated bacteria was observed. The number of bacterial species in urine did not correlate with blood parameters. The presence of leukocytes (p = 0.013) and proteins (p = 0.004) in urine was related to a greater variety of bacteria found in urine specimens. (4) Conclusions: We obtained a similar spectrum of bacteria from the initial and middle urine streams. We also showed that there is a change in bacteria species affected by the treatment of prostate cancer patients, with both antibiotics before gold fiducial implantation and radiotherapy.

Isolation and Identification of Lactococcus lactis and Weissella cibaria Strains from Fermented Beetroot and an Investigation of Their Properties as Potential Starter Cultures and Probiotics.

The presence of certain microorganisms in dairy products or silage is highly desirable. Among them are probiotic strains of lactic acid bacteria (LAB), which show many beneficial features, including antimicrobial properties that support the development of beneficial microflora; in addition, owing to their biochemical activity, they influence the nutritional, dietary, and organoleptic properties of food products. Before being placed on the market, each strain requires separate testing to determine its probiotic properties and effectiveness. The aim of this study was to isolate LAB strains from a pickled beetroot sample that could be used in the dairy industry and with the potential to be considered as a probiotic in the future. Two strains identified using the MALDI technique were selected-Lactococcus lactis and Weissella cibaria. The optimal growth conditions of the strains were determined, and their proteolytic properties were assessed with the use of the o-PA reagent and spectrophotometry. The lipid profile was analyzed using the SALDI (surface-assisted laser desorption/ionization) technique and silver nanoparticles. High-performance liquid chromatography was used to assess the ability of the strains to synthesize beneficial metabolites, such as B vitamins (B2, B3, and B9) or lactic acid, and gas chromatography was used to analyze the substances responsible for organoleptic properties. Moreover, the ability to inhibit the growth of pathogenic strains was also tested in the selected strains. Both tested strains demonstrated the desired properties of starter cultures for future use in functional food production, showing that fermented plant products can serve as valuable potential probiotic sources.

New sources of lactic acid bacteria with potential antibacterial properties.

In the face of the growing demand for functional food, the search for new sources of lactic acid bacteria (LAB) becomes a priority. In our research, we used multiplied culture conditions followed by identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry for seeking LAB strains in plant- and animal-derived sources. Furthermore, the selected LAB isolates were examined for their proteolytic activity as well as antimicrobial action against different bacterial pathogens. The applied method appeared to be useful tool for searching LAB strains within different types of the biological matrices. The best source of the LABs was from calf. Comparing properties of the two selected LABs, those isolated from calf demonstrated the greatest proteolytic and antibacterial properties suggesting that gastrointestinal microbiota are the most valuable LAB source. Nevertheless, second selected strain derived from pickled cucumber juice may be also treated as a promising source of potential probiotic strains.

Capillary Zone Electrophoresis in Tandem with Flow Cytometry in Viability Study of Various ATCC Bacterial Strains under Antibiotic Treatment.

The aim of this study was to develop an innovative method of examining bacterial survival using capillary zone electrophoresis (CZE) and flow cytometry (FC) as a reference method. For this purpose, standard strains of bacteria from the ATCC collection were used: Enterococcus faecalis ATCC 14506, Staphylococcus aureus ATCC 11632, Klebsiella pneumoniae ATCC 10031, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922, as well as seven antibiotics with different antimicrobial mechanisms of action. The ratio of live and dead cells in the tested sample in CZE measurements were calculated using our algorithm that takes into account the detection time. Results showed a high agreement between CZE and FC in the assessment of the percentage of live cells exposed to the stress factor in both antibiotic susceptibility and time-dependent assays. The applied measuring system to assess the effectiveness of antibiotic therapy in in vitro conditions is a method with great potential, and the data obtained with the use of CZE mostly correspond to the expected drug sensitivity according to EUCAST and CLSI guidelines.

A new approach to identifying pathogens, with particular regard to viruses, based on capillary electrophoresis and other analytical techniques.

Fast determination, identification and characterization of pathogens is a significant challenge in many fields, from industry to medicine. Standard approaches (e.g., culture media and biochemical tests) are known to be very time-consuming and labor-intensive. Conversely, screening techniques demand a quick and low-cost grouping of microbial isolates, and current analysis call for broad reports of pathogens, involving the application of molecular, microscopy, and electromigration techniques, DNA fingerprinting and also MALDI-TOF methods. The present COVID-19 pandemic is a crisis that affects rich and poor countries alike. Detection of SARS-CoV-2 in patient samples is a critical tool for monitoring disease spread, guiding therapeutic decisions and devising social distancing protocols. The goal of this review is to present an innovative methodology based on preparative separation of pathogens by electromigration techniques in combination with simultaneous analysis of the proteome, lipidome, and genome using laser desorption/ionization analysis.

Culturomics Approach to Identify Diabetic Foot Infection Bacteria.

The main goal of the study was to evaluate the usefulness of the culturomics approach in the reflection of diabetic foot infections (DFIs) microbial compositions in Poland. Superficial swab samples of 16 diabetic foot infection patients (Provincial Polyclinical Hospital in Torun, Poland) were subjected to culturing using 10 different types of media followed by the identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Biotyper platform. Identified 204 bacterial isolates representing 18 different species-mostly Enterococcus faecalis (63%) and Staphylococcus aureus (44%). Most of the infections (81%) demonstrated a polymicrobial character. Great differences in the species coverage, the number of isolated Gram-positive and Gram-negative bacteria, and the efficiency of the microbial composition reflection between the investigated media were revealed. The use of commonly recommended blood agar allowed to reveal only 53% of the entire microbial composition of the diabetic foot infection samples, which considerably improved when the chromagar orientation and vancomycin-resistant enterococi agar were applied. In general, efficiency increased in the following order: selective < universal < enriched < differential media. Performed analysis also revealed the impact of the culture media composition on the molecular profiles of some bacterial species, such as Corynebacterium striatum, Proteus mirabilis or Morganella morganii that contributed to the differences in the identification quality. Our results indicated that the culturomics approach can significantly improve the accuracy of the reflection of the diabetic foot infections microbial compositions as long as an appropriate media set is selected. The chromagar orientation and vancomycin-resistant enterococi agar media which were used for the first time to study diabetic foot infection microbial profiles demonstrate the highest utility in the culturomics approach and should be included in further studies directed to find a faster and more reliable diabetic foot infection diagnostic tool.

Study on Molecular Profiles of Staphylococcus aureus Strains: Spectrometric Approach.

Staphylococcus aureus remains a major health problem responsible for many epidemic outbreaks. Therefore, the development of efficient and rapid methods for studying molecular profiles of S. aureus strains for its further typing is in high demand. Among many techniques, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) represents a timely, cost-effective, and reliable strain typing approach, which is still rarely used due to insufficient knowledge about the impact of sample preparation and analysis conditions on the molecular profiles and strain classification efficiency of S. aureus. The aim of this study was to evaluate the effect of the culture conditions and matrix type on the differentiation of molecular profiles of various S. aureus strains via the MALDI TOF MS analysis and different computational methods. The analysis revealed that by changing the culture conditions, matrix type, as well as a statistical method, the differentiation of S. aureus strains can be significantly improved. Therefore, to accelerate the incorporation of the MALDI-based strain typing in routine laboratories, further studies on the standardization and searching of optimal conditions on a larger number of isolates and bacterial species are of great need.

Orodispersible films and tablets with prednisolone microparticles.

Orodispersible tablets (ODTs) and orodispersible films (ODFs) are solid oral dosage forms disintegrating or dissolving rapidly when placed in the mouth. One of the main issues related to their preparation is an efficient taste masking of a bitter drug substance. Therefore, the aim of this study was to prepare and evaluate the microparticles intended to mask a bitter taste of the prednisolone and use them in further preparation of two orodispersible dosage forms. Microparticles based on the Eudragit E PO or E 100 as a taste-masking agent were prepared with spray-drying technique. Tablets containing microparticles, co-processed ODT excipient Pharmaburst, and lubricant were directly compressed with single-punch tablet press. Orodispersible films were prepared by casting polymeric solutions of hydroxypropyl methylcellulose containing uniformly dispersed microparticles. Physicochemical properties of microparticles were evaluated, as well as mechanical properties analysis, disintegration time measurements and dissolution tests were performed for prepared dosage forms. Both formulations showed good mechanical resistance while maintaining excellent disintegration properties. The dissolution studies showed good masking properties of microparticles with Eudragit E 100. The amount of prednisolone released during the first minute in phosphate buffer 6.8 was around 0.1%. After incorporation into the orodispersible forms, the amount of released prednisolone increased significantly. It was probably the effect of faster microparticles wetting in orodispersible forms and their partial destruction by compression force during tableting process.