Influence of supplementation with iron and probiotic bacteria Lactobacillus plantarum and Lactobacillus curvatus on selected parameters of inflammatory state in rats on a high-fat iron-deficient diet.

BACKGROUND: A high-fat (HF) diet, diet iron deficiency and iron supplementation may affect inflammatory parameters. Probiotics influence both iron metabolism and inflammation. We compared the inflammatory state in rats on a HF iron-deficient diet receiving oral iron, Lactobacillus plantarum and Lactobacillus curvatus in different combinations. METHODS: This was a two-stage experiment. In groups C (n = 8) and HF (n = 8), rats ate a control or HF diet, respectively, for 16 weeks. In the group HFDEF (n = 48), rats ate a HF iron-deficient diet for 8 weeks (first stage) and were subsequently divided into 6 groups (n = 8 each) receiving the following for a further 8 weeks (second stage): HFDEF - a HF iron-deficient diet; HFDEFFe - a HF iron-deficient diet with iron; HFDEFLp and HFDEFLc - a HF iron-deficient diet with L. plantarum or L. curvatus, respectively; and HFDEFFeLp and HFDEFFeLc - a HF iron-deficient diet with iron and L. plantarum or L. curvatus, respectively. Body composition analysis and blood sampling was performed. Markers of iron status and levels of total antioxidant status (TAS), C-reactive protein (CRP), tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were measured in the blood. RESULTS: TAS was higher in the HFDEF group (756.57 ± 489.53 ng mL-1) versus the HFDEFLc group (187.04 ± 47.84 ng mL-1; P = 0.022). No more differences were found between groups, or in TAS, CRP, TNF-α and IL-6 concentrations. Also, no differences were found between groups for alanine and aspartate aminotransferases, glucose, total cholesterol, low- and high-density lipoproteins and triglycerides. TAS level was positively correlated with ferritin concentration, IL-6 with TAS and TNF-α with hepcidin level. CONCLUSIONS: Supplementation with L. plantarum, L. curvatus and iron in combinations exerts no influence on inflammatory status, lipid profile, hepatic function and serum fasting glucose in rats on a HF iron-deficient diet. © 2024 Society of Chemical Industry.

Listeria monocytogenes Isolates from Meat Products and Processing Environment in Poland Are Sensitive to Commonly Used Antibiotics, with Rare Cases of Reduced Sensitivity to Ciprofloxacin.

Antibiotic resistance is a global health problem, causing not only an increased mortality rate of bacterial infections but also economic losses due to, among other reasons, the need for longer hospital stays. Listeria monocytogenes is one of the foodborne pathogens with the ability to induce a serious illness called listeriosis, with approximately 20-30% fatal outcomes. The treatment regimen of listeriosis in humans includes the administration of antibiotics (in most cases, ampicillin or trimethoprim with sulfamethoxazole in case of allergies to ß-lactams), so the resistance of this pathogen to antibiotics can potentially lead to increased mortality. The antibiotic sensitivity status of n = 153 L. monocytogenes isolates originating from meat food samples (raw and processed) and meat-processing environment (both contacting and non-contacting with food) collected between October 2020 and November 2021 in Poland was examined in this study. Susceptibility to antibiotics was determined using the disc diffusion method on Mueller-Hinton agar plates. All collected samples were susceptible to 9 antibiotics: ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), penicillin (10 IU), streptomycin (10 µg), sulfamethoxazole/trimethoprim (1.25/23.75 µg), tetracycline (30 µg) and vancomycin (30 µg). Some of the isolates (n = 10; 6.5%) showed reduced susceptibility to ciprofloxacin (5 µg), which was classified as an intermediate response. All these ten isolates were collected from surfaces contacting with food in food-processing facilities.

Circulating Microbial Cell-Free DNA in Health and Disease.

Human blood contains low biomass of circulating microbial cell-free DNA (cfmDNA) that predominantly originates from bacteria. Numerous studies have detected circulating cfmDNA in patients with infectious and non-infectious diseases, and in healthy individuals. Remarkable differences were found in the microbial composition of healthy subjects and patients compared to cohorts with various diseases or even patients with diversified prognoses, implying that these alterations may be associated with disease development. Although the function of circulating cfmDNA needs to be elucidated (whether it acts as a bystander of dysbiosis or a key player in disease development), several studies have demonstrated its potential as a non-invasive biomarker that may improve diagnosis and treatment efficacy. The origin of circulating cfmDNA is still the subject of much deliberation, but studies have identified members of various microbiome niches, including the gut, oral cavity, airways, and skin. Further studies investigating the origin and function of circulating cfmDNA are needed. Moreover, low-biomass microbiome studies are prone to contamination, therefore stringent negative experimental control reactions and decontamination frameworks are advised in order to detect genuine circulating cfmDNA.

A Clinical Outcome of the Anti-PD-1 Therapy of Melanoma in Polish Patients Is Mediated by Population-Specific Gut Microbiome Composition.

The gut microbiota is considered a key player modulating the efficacy of immune checkpoint inhibitor therapy. The study investigated the association between the response to anti-PD-1 therapy and the baseline gut microbiome in a Polish cohort of melanoma patients, alongside selected agents modifying the microbiome. Sixty-four melanoma patients enrolled for the anti-PD-1 therapy, and ten healthy subjects were recruited. The response to the treatment was assessed according to the response evaluation criteria in solid tumors, and patients were classified as responders or non-responders. The association between selected extrinsic factors and response was investigated using questionnaire-based analysis and the metataxonomics of the microbiota. In the responders, the Bacteroidota to Firmicutes ratio was higher, and the richness was decreased. The abundance of Prevotella copri and Bacteroides uniformis was related to the response, whereas the non-responders' gut microbiota was enriched with Faecalibacterium prausnitzii and Desulfovibrio intestinalis and some unclassified Firmicutes. Dietary patterns, including plant, dairy, and fat consumption as well as gastrointestinal tract functioning were significantly associated with the therapeutic effects of the therapy. The specific gut microbiota along with diet were found to be associated with the response to the therapy in the population of melanoma patients.

Nonhemolytic Listeria monocytogenes-Prevalence Rate, Reasons Underlying Atypical Phenotype, and Methods for Accurate Hemolysis Assessment.

Listeria monocytogenes is a foodborne pathogen that typically presents ß-hemolytic activity. However, there are literature reports indicating that L. monocytogenes strains are sometimes nonhemolytic or their zones of hemolysis are perceivable only after removal of the colonies from the agar plate. Nonhemolytic L. monocytogenes are most commonly encountered in food products, but some have also been detected in clinical samples. Usually, atypical bacteria of this species belong to serotype 1/2a. Mutations of the prfA gene sequence are the most common reason for changed phenotype, and mutations of the hly gene are the second most common cause. There are also reports that the methodology used for detecting hemolysis may influence the results. Sheep or horse blood, although most commonly used in modern studies, may not allow for the production of clear hemolytic zones on blood agar, whereas other types of blood (guinea pig, rabbit, piglet, and human) are more suitable according to some studies. Furthermore, the standard blood agar plate technique is less sensitive than its modifications such as bilayer or top-layer (overlay) techniques. The microplate technique (employing erythrocyte suspensions) is probably the most informative when assessing listerial hemolysis and is the least susceptible to subjective interpretation.

Natural Plant-Derived Chemical Compounds as Listeria monocytogenes Inhibitors In Vitro and in Food Model Systems.

Listeria monocytogenes is a foodborne pathogen, sporadically present in various food product groups. An illness caused by the pathogen, named listeriosis, has high fatality rates. Even though L. monocytogenes is resistant to many environmental factors, e.g., low temperatures, low pH and high salinity, it is susceptible to various natural plant-derived antimicrobials (NPDA), including thymol, carvacrol, eugenol, trans-cinnamaldehyde, carvone S, linalool, citral, (E)-2-hexenal and many others. This review focuses on identifying NPDAs active against L. monocytogenes and their mechanisms of action against the pathogen, as well as on studies that showed antimicrobial action of the compounds against the pathogen in food model systems. Synergistic action of NDPA with other factors, biofilm inhibition and alternative delivery systems (encapsulation and active films) of the compounds tested against L. monocytogenes are also summarized briefly.

Secretory IgA in Intestinal Mucosal Secretions as an Adaptive Barrier against Microbial Cells.

Secretory IgA (SIgA) is the dominant antibody class in mucosal secretions. The majority of plasma cells producing IgA are located within mucosal membranes lining the intestines. SIgA protects against the adhesion of pathogens and their penetration into the intestinal barrier. Moreover, SIgA regulates gut microbiota composition and provides intestinal homeostasis. In this review, we present mechanisms of SIgA generation: T cell-dependent and -independent; in different non-organized and organized lymphoid structures in intestinal lamina propria (i.e., Peyer's patches and isolated lymphoid follicles). We also summarize recent advances in understanding of SIgA functions in intestinal mucosal secretions with focus on its role in regulating gut microbiota composition and generation of tolerogenic responses toward its members.

Effectiveness of Phage-Based Inhibition of Listeria monocytogenes in Food Products and Food Processing Environments.

Providing safe products and compliance of legal requirements is still a great challenge for food manufacturers regarding microbiological safety, especially in the context of Listeria monocytogenes food contamination. L. monocytogenes is a human pathogen, which, due to the ability of survival and proliferation in preservation conditions such as high salinity, acidity and refrigeration temperatures, is a significant threat to the food industry. Novel methods of elimination of the bacterial pathogen in food products and food processing environments are required. Among emerging technologies, one of the very promising solutions is using bacteriophages as natural control agents. This review focus on the major aspects of phage-based inhibition of L. monocytogenes in aspects of food safety. We describe an overview of foods and technological factors influencing the efficacy of phage use in biocontrol of L. monocytogenes. The most noteworthy are food matrix properties, phage concentration and stability, the time of phage application and product storage temperature. The combined methods, phage immobilization (active packing), pathogen resistance to phages and legislation aspects of antilisterial bacteriophage use in the food industry are also discussed.

Lactobacillus spp. belonging to the Casei group display a variety of adhesins.

BACKGROUND: n. Adhesion of bacteria from the genus Lactobacillus to the gastrointestinal epithelium is, to a considerable degree, dependent on the interactions between adhesins found on the surface of bacterial cells and elements found within the epithelium. A significant role in these interactions is played by bacterial pro teins exposed to the cell wall surface, which are capable of binding to molecules of substances comprising the extracellular matrix of the intestinal epithelium. METHODS: In order to analyze the extracellular proteome of intestinal bacteria in terms of the presence of cell adhesion molecules, a total of twenty strains from the Lactobacillus spp. group Casei were tested. The analyses were conducted using SDS PAGE, 2-D electrophoresis, Western blot and mass spectrometry. An experiment was also conducted to assess the adhesion capacity of the tested strains to cervical epithelial cells (HeLa). RESULTS: The tested strains varied in their adhesion efficiency to HeLa cells, ranging from 0.5% to 29%. Us- ing electrophoretic methods a total of 54 extracellular protein fractions were distinguished in these strains, additionally identifying potential adhesion molecules (e.g. a surface antigen of the NLP/P60 family and a small heat shock protein/chaperonin). CONCLUSIONS: The identification of these proteins in the extracellular proteome of Latobacillus spp. isolates may suggest that they serve currently unknown functions on the cell surface, including those connected with the interactions between bacteria and the intestinal epithelium. Such analyses may provide insight into new factors promoting probiotic adhesion to various types of epithelial cells.

Gut microbiota in cirrhotic liver transplant candidates.

BACKGROUND/AIMS: The purpose of this study was to evaluate the gut microflora of liver transplant candidates. METHODOLOGY: Fecal microflora of 20 cirrhotic liver transplant candidates was analyzed basing on prospectively collected stool samples. The results were compared with those of 20 non-cirrhotic patients with liver disease and/or abnormal liver function tests, 20 patients with Crohn's disease, and 20 patients without any gastrointestinal disease. Moreover, correlations between particular counts of microbiota, as well as between microbial counts and stool pH were examined. RESULTS: The pattern of fecal microbiota of liver transplant candidates was characterized by increased counts of lactobacilli (p=0.001), including hydrogen peroxide producing strains (p=0.008). In these patients, lactobacilli were positively correlated to enterococci (p=0.006) and bifidobacteria (p=0.004). No correlations other than those observed for lactobacilli in general were observed between hydrogen peroxide producing lactobacilli and the remaining microbiota. Increased yeast and Escherichia coli counts were associated with a tendency towards lower (p=0.095) and higher (p=0.072) stool pH, respectively. CONCLUSIONS: Surprisingly, gut microflora of liver transplant candidates with cirrhosis is particularly enriched with lactobacilli, including hydrogen peroxide producing strains. Thus, the use of other potentially beneficial microorganisms, such as particular yeast strains, might be more appropriate for these patients.

Expression of bacteriocin divercin AS7 in Escherichia coli and its functional analysis.

Bacteriocins are small peptides with antimicrobial activity, that are produced by bacteria. Four classes of bacteriocins produced by lactic acid bacteria have been defined. Class IIa bacteriocins are promising candidates for industrial applications due to their high biological activity and their physicochemical properties. Divercin AS7 is a class IIa bacteriocin produced by Carnobacterium divergens AS7. It shows antibacterial activity against pathogens and food spoilage flora including Listeria spp. Little is known about the impact of class IIa bacteriocins upon eukaryotic cells. The safe use of bacteriocins as food biopreservatives requires the absence of cytotoxicity to human cells. To analyze the impact of divercin AS7 on human enterocytes, we expressed the recombinant divercin AS7 in the Escherichia coli BL21DE3pLys strain and conducted in vitro studies to evaluate the safety of recombinant divercin AS7. No cytotoxic effect on differentiated monolayer Caco-2 cells and no apoptotic appearance were observed when recombinant divercin AS7 was used at a concentration of 2 µg ml-1. In our study, divercin AS7 also did not interfere with differentiated Caco-2 cells monolayer integrity. The obtained results suggest that divercin AS7 is a promising peptide for the food industry.

Influence of phenolic acids on indole acetic acid production and on the type III secretion system gene transcription in food-associated Pseudomonas fluorescens KM05.

The purpose of these investigations was to evaluate the reduction capability of phenolic acids (ferulic, chlorogenic, gallic, and p-coumaric acids) on indole acetic acid synthesis by food-associated Pseudomonas fluorescens KM05. Specific genetic primer for the type III secretion system (TTSS) in P. fluorescens KM05 was designed and the influence of phenolic acids on its expression was investigated. In the work the ferulic and chlorogenic acids at the concentration of 0.02 and 0.04 µg/ml affected on bacterial growth pattern and the signal molecules production. The phenolic acids, that were appreciable effective against P. fluorescens KM05 indole acetic acid production, significantly suppressed TTSS gene.

SpaCBA sequence instability and its relationship to the adhesion efficiency of Lactobacillus casei group isolates to Caco-2 cells.

The ability to adhere to enterocytes is one of the key features of probiotics. This process involves a number of factors, among which the important role of pili was demonstrated. Some Lactobacillus species are confirmed to have heterotrimeric spaCBA type pili. The aim of this study was to identify spaCBA pili in strains of selected Lactobacillus spp. and assess the impact of their presence and sequence polymorphism on the adhesion of these strains to enterocytes. Total 20 bacterial strains of L. rhamnosus, L. casei and L. paracasei were tested. The presence of pilus specific proteins coding genes spaA, spaB and spaC was verified by PCR in order to identify the presence of sequence polymorphism in the genes possibly affecting the structure of the spaCBA pilus. To correlate spaCBA polymorphism to adhesion capability the adhesion assay was carried out using Caco-2 cell line. The effectiveness of the adhesion was measured using a scintillation counter. The Lactobacillus strains analyzed showed the adhesion to Caco-2 enterocytes capability from 0.6% to 19.6%. The presence of spaCBA pili is a factor increasing the adhesion efficiency of Lactobacillus spp. to Caco-2 enterocytes. Lack of these structures on the surface of bacterial cells results in the reduction in adhesion efficiency, indicating its important role in the adhesion process. But not in all cases the correlation between the presence of protein spaCBA structures and adhesion efficiency was observed, what may indicate the important role of other factors in adhesion of analyzed strains to Caco-2 cells.

Conversion of glycerol to 1,3-propanediol by Citrobacter freundii and Hafnia alvei - newly isolated strains from the Enterobacteriaceae.

In this study, nearly 4000 bacterial strains from the family of Enterobacteriaceae isolated from different environments were screened for ability to convert glycerol to 1,3-propanediol (1,3-PD). The aim of the research was to isolate 1,3-PD producers from the natural environment, identify and characterize the best isolates. Three selective media were tested to usefulness in the isolation of bacteria from the family Enterobacteriaceae. Only, 28% of examined isolates could synthesize 1,3-PD from glycerol. 1,3-PD producing bacteria were identified by API 20E tests and 16S rRNA sequences to be Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii and Hafnia alvei. It is the first time, when the fermentation glycerol to 1,3-PD by H. alvei was investigated. The selected strains (C. freundii AD119 and H. alvei AD27) were analyzed on a bioreactor scale under constant pH value 7.0 at temperature of 30°C and 37°C. After 40h in batch fermentation, H. alvei AD27 produced 11.3g/L of 1,3-PD at 37°C. For C. freundii AD119, the best results were obtained at temperature of 30°C. After 24h of fermentation, the 1,3-PD concentration reached above 23 g/L of 1,3-PD.

[The NICE system as a tool for protein expression in Lactococcus lactis]. / Charakterystyka systemu nizynowego jako narzedzia do produkcji bialek i peptydów w komórkach Lactococcus lactis.

The progress in genetic engineering allows to employ still growing number of bacterial strains for recombinant gene expression. Among them are many lactic acid bacteria including Lactococcus lactis which belongs to the most developed ones. Those microorganisms are ideal for heterologous protein expression because they can synthesize nisin which is a bacteriocin that can be used as an inductor factor. The recently developed NICE system is molecular tool that allows to produce heterolguos proteins in Lactococcus lactis. The NICE system needs three components for correct operation: host strain, plasmid and inductor factor - nisin. The NICE system proved to be valuable tool for recombinant protein expression including synthesis of heterologous antimicrobial peptides, membrane proteins, and vaccines development.

Isolation process of industrially useful Clostridium bifermentans from natural samples.

A selective isolation procedure of clostridial strains from natural samples able to convert glycerol to 1,3-propanediol (1,3-PD) and organic acids was investigated. The modified PY medium of high concentration of NaHCO(3) was shown to be highly selective for Clostridium bifermentans. Obtained isolates produced mainly 1,3-PD, lactic, acetic, and formic acids from glycerol.

Impact of DNA microarray data transformation on gene expression analysis - comparison of two normalization methods.

Two-color DNA microarrays are commonly used for the analysis of global gene expression. They provide information on relative abundance of thousands of mRNAs. However, the generated data need to be normalized to minimize systematic variations so that biologically significant differences can be more easily identified. A large number of normalization procedures have been proposed and many softwares for microarray data analysis are available. Here, we have applied two normalization methods (median and loess) from two packages of microarray data analysis softwares. They were examined using a sample data set. We found that the number of genes identified as differentially expressed varied significantly depending on the method applied. The obtained results, i.e. lists of differentially expressed genes, were consistent only when we used median normalization methods. Loess normalization implemented in the two software packages provided less coherent and for some probes even contradictory results. In general, our results provide an additional piece of evidence that the normalization method can profoundly influence final results of DNA microarray-based analysis. The impact of the normalization method depends greatly on the algorithm employed. Consequently, the normalization procedure must be carefully considered and optimized for each individual data set.

Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells.

Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.

Biodegradation and surfactant-mediated biodegradation of diesel fuel by 218 microbial consortia are not correlated to cell surface hydrophobicity.

In this study, we elucidated the role of cell surface hydrophobicity (microbial adhesion to hydrocarbons method, MATH) and the effect of anionic rhamnolipids and nonionic Triton X-100 surfactants on biodegradation of diesel fuel employing 218 microbial consortia isolated from petroleum-contaminated soils. Applied enrichment procedure with floating diesel fuel as a sole carbon source in liquid cultures resulted in consortia of varying biodegradation potential and diametrically different cell surface properties, suggesting that cell surface hydrophobicity is a conserved parameter. Surprisingly, no correlations between cell surface hydrophobicity and biodegradation of diesel fuel were found. Nevertheless, both surfactants altered cell surface hydrophobicity of the consortia in similar manner: increased for the hydrophilic and decreased for the hydrophobic cultures. In addition to this, the surfactants exhibited similar influence on diesel fuel biodegradation: Increase was observed for initially slow-degrading cultures and the opposite for fast degraders. This indicates that in the surfactant-mediated biodegradation, effectiveness of surfactants depends on the specification of microorganisms and not on the type of surfactant. In contrary to what was previously reported for pure strains, cell surface hydrophobicity, as determined by MATH, is not a good descriptor of biodegrading potential for mixed cultures.

Biodegradation of diesel fuel by a microbial consortium in the presence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues.

Fast development of ionic liquids as gaining more and more attention valuable chemicals will undoubtedly lead to environmental pollution. New formulations and application of ionic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We hypothesize that in the presence of diesel fuel low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side-chain length from C(3) to C(18)) on biodegradation of diesel fuel by a bacterial consortium was investigated. Whereas test performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher homologues (C(8)-C(18)) caused a decrease in diesel fuel biodegradation. As a result of exposure to toxic compounds also modification in cell surface hydrophobicity was observed (MATH). Disulphine blue active substances method was employed to determine partitioning index of ionic liquids between water and diesel fuel phase, which varied from 1.1 to 51% for C(3) and C(18) homologues, respectively. We conclude that in the presence of hydrocarbons acting as a solvent, the increased bioavailability of hydrophobic homologues is responsible for the decrease in biodegradation efficiency of diesel fuel.