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Magdalena Strus,1 Ewa Helwich,2 Ryszard Lauterbach,3 Beata Rzepecka-Węglarz,4 Katarzyna Nowicka,2 Maria Wilińska,5 Jerzy Szczapa,6 Małgorzata Rudnicka,7 Helena Sławska,8 Marek Szczepański,9 Aneta Waśko,10 Aleksandra Mikołajczyk-Cichońska,10 Anna Tomusiak-Plebanek,1 Piotr B Heczko1 1Department of Microbiology, Jagiellonian University Medical College, Kraków, Poland; 2Department of Neonatology, Institute of Mother and Child, Warszawa, Poland; 3Clinical Department of Neonatology, University Hospital, Kraków, Poland; 4Department of Neonatal Intensive Care, “UJASTEK” Medical Centre, Kraków, Poland; 5Clinical Department of Neonatology, Independent Public Clinical Hospital CMKP, Warszawa, Poland; 6Department of Neonatology, Gynecology and Obstetrics Clinical Hospital, Poznań, Poland; 7Department of Neonatology, Regional Specialist Hospital, Wrocław, Poland; 8Department of Neonatology, Specialist Hospital No. 2, Bytom, Poland; 9Clinic Department of Neonatology and Neonatal Intensive Care, University Clinical Hospital, Białystok, Poland; 10Medical Department, IBSS BIOMED S.A., Kraków, Poland Aim: Probiotic bacteria administered directly after birth to preterm neonates may improve gastrointestinal function and may reduce the incidence of late-onset sepsis, which is a frequent complication in this group. Purpose: The main objective of this study was to evaluate whether a new probiotic bacterial mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 given to preterm, low-birth-weight neonates would influence composition of their gut microbiota and sepsis rates. Patients and methods: This study was a multicenter, randomized, double-blind, placebo-controlled trial conducted in clinical centers of neonatal care in Poland. A probiotic or placebo preparation was given twice daily to 181 preterm low-birth-weight neonates who were eligible for enteral feeding between July 2012 and July 2013. The probiotic was given to 90 neonates, while placebo was given to 91 neonates. The gut microbiota was monitored by microbiological analysis of stool samples. Sepsis episodes were detected on the basis of clinical and laboratory findings and confirmed by blood cultures. Results: Tested probiotic administration resulted in continuous increase of the Lactobacillus and Bifidobacterium counts in the gut microbiota. The applied tested strains successfully colonized the neonates gut since they were present in over 90% of stool samples, which was confirmed by molecular analysis. Regardless of the study group (probiotic or placebo), B. breve ­colonization correlated with lower staphylococcal sepsis incidence, which was irrespective of whether ­probiotics were given. No sepsis case caused by strains included in study probiotic was recorded. Conclusion: Appropriately selected and characterized probiotic bacteria may be safely given to preterm neonates to normalize their distorted gut microbiota and may contribute to lower staphylococcal sepsis rates. Keywords: probiotics, LBW neonates, staphylococcal sepsis, gut microbiota, Lactobacillus, Bifidobacterium

Anna Tomusiak-Plebanek,1 Piotr Heczko,1 Beata Skowron,2 Agnieszka Baranowska,2 Krzysztof Okoń,3 Piotr J Thor,2 Magdalena Strus1 1Department of Microbiology, Jagiellonian University Medical College, Poland; 2Department of Pathophysiology, Jagiellonian University Medical College, Poland; 3Department of Pathomorphology, Jagiellonian University Medical College, Poland Purpose: Some lactobacilli, which possess superoxide dismutase-like activity and catalase activity naturally, have strong antioxidative properties. The aim of this study was to identify such strains and check which of them play a crucial role in alleviating intestinal inflammation. Methods: We selected two Lactobacillus strains for use in animal studies: L. plantarum 30B (which has the highest catalase activity) and L. acidophilus 900 (which has the highest dismutase-like activity). Forty mice (C57B1/6J) were divided into four experimental groups with ten mice in each group. Group I (control group) was not supplemented with Lactobacillus, group II (catalase group) was orally supplemented with L. plantarum 30B, group III (dismutase-like group) was supplemented with L. acidophilus 900, and group IV (mixed group) was supplemented with both Lactobacillus strains. For 23 days, the temperature and body mass of each mouse were recorded and fecal samples for microbiological examination were collected. On day 23, the animals were sacrificed, and their intestines were removed for microbiological and histopathological studies. Results: Compared to the control group, the highest drop in the body temperature was observed in groups II (P

Anna Tomusiak,1 Magdalena Strus,1 Piotr B Heczko,1 Paweł Adamski,2 Grzegorz Stefański,3 Aleksandra Mikołajczyk-Cichońska,3 Magdalena Suda-Szczurek3 1Department of Microbiology, Jagiellonian University Medical College, 2Institute of Nature Conservation, Polish Academy of Sciences, 3IBSS BIOMED SA, Kraków, Poland Objective: The main objective of this study was to evaluate whether vaginal administration of probiotic Lactobacillus results in their colonization and persistence in the vagina and whether Lactobacillus colonization promotes normalization and maintenance of pH and Nugent score. Patients and methods: The study was a multicenter, randomized, double-blind, and placebo-controlled trial. Altogether, 376 women were assessed for eligibility, and signed informed consent. One hundred and sixty eligible women with abnormal, also called intermediate, vaginal microflora, as indicated by a Nugent score of 4–6 and pH >4.5 and zero or low Lactobacillus count, were randomized. Each participant was examined four times during the study. Women were randomly allocated to receive either the probiotic preparation inVag®, or a placebo (one capsule for seven consecutive days vaginally). The product inVag includes the probiotic strains Lactobacillus fermentum 57A, Lactobacillus plantarum 57B, and Lactobacillus gasseri 57C. We took vaginal swabs during visits I, III, and IV to determine the presence and abundance of bacteria from the Lactobacillus genus, measure the pH, and estimate the Nugent score. Drug safety evaluation was based on analysis of the types and occurrence of adverse events. Results: Administration of inVag contributed to a significant decrease (between visits) in both vaginal pH (P

Abstract Background An important aspect in the microbiology of the reproductive system of small animals is the potential occurrence of probiotic bacteria, such as lactic acid bacteria (LAB) of the genus Lactobacillus. The presence of these microorganisms is significant due to their strong antibacterial and antifungal properties. This study aimed to select probiotic strains from the oral cavity and vagina that have outstanding antibacterial properties against typical genital pathogens of the female dog reproductive tract. Results The antagonistic activity of ten LAB strains was tested against seven etiological agents isolated from the genital tract of female dogs with signs of inflammation. LAB strains with the greatest ability to inhibit the growth of indicator bacteria were Lactobacillus plantarum and L. acidophilus, while L. fermentum and L. brevis strains inhibited growth the least. Almost all strains showed a complete lack of adherence to Caco-2 epithelial cells. Conclusions All tested LAB isolates inhibited the in vitro growth of either Gram-positive or Gram-negative pathogens, suggesting that potential probiotic strains could contribute to the balance of the normal vaginal microbiota. Furthermore, they could be considered for use as prophylactic agents or as an alternative to antibiotic therapy for infections in dogs.

OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with β-glucan might improve their response to P. aeruginosa infections., Methods: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae β-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo β-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared., Results: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced., Conclusions: Training of murine macrophages with S. cerevisiae β-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such β-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation., (© 2024. The Author(s).)

Introduction: Biofilm-associated infections persist as a therapeutic challenge in contemporary medicine. The efficacy of antibiotic therapies is ineffective in numerous instances, necessitating a heightened focus on exploring novel anti-biofilm medical strategies. Among these, iminosugars emerge as a distinctive class of compounds displaying promising biofilm inhibition properties., Methods: This study employs an in vivo wound infection mouse model to evaluate the effectiveness of PDIA in treating biofilm-associated skin wound infections caused by Staphylococcus aureus and Pseudomonas aeruginosa . Dermic wounds in mice were infected with biofilm-forming strains, specifically S. aureus 48 and P. aeruginosa 5, which were isolated from patients with diabetic foot, and are well-known for their strong biofilm formation. The subsequent analysis included clinical, microbiological, and histopathological parameters. Furthermore, an exploration into the susceptibility of the infectious strains to hydrogen peroxide was conducted, acknowledging its potential presence during induced inflammation in mouse dermal wounds within an in vivo model., Results: The findings revealed the efficacy of PDIA iminosugar against the S. aureus strain, evidenced by a reduction in bacterial numbers within the wound and the inflammatory focus., Discussion: This study suggests that PDIA iminosugar emerges as an active and potentially effective antibiofilm agent, positioning it as a viable treatment option for staphylococcal infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kozień, Policht, Heczko, Arent, Bracha, Pardyak, Pietsch-Fulbiszewska, Gallienne, Piwowar, Okoń, Tomusiak-Plebanek and Strus.)

Colorectal cancer (CRC) is one of the foremost causes of cancer-related deaths. Lately, a close connection between the course of CRC and the intestinal microbiota has been revealed. Vitamin K 2 (VK 2 ) is a bacterially derived compound that plays a crucial role in the human body. Its significant anti-cancer properties may result, inter alia, from a quinone ring possessing a specific chemical structure found in many chemotherapeutics. VK 2 can be supplied to our body exogenously, i.e., through dietary supplements or fermented food (e.g., yellow cheese, fermented soybeans -Natto), and endogenously, i.e., through the production of bacteria that constantly colonize the human microbiome of the large intestine.This paper focuses on endogenous K 2 synthesized by the most active members of the human gut microbiome. This analysis tested 86 intestinally derived bacterial strains, among which the largest VK 2 producers (Lactobacillus, Bifidobacterium, Bacillus) were selected. Moreover, based on the chosen VK 2 -MK4 homolog, the potential of VK 2 penetration into Caco-2 cells in an aqueous environment without the coexistence of fats, pancreatic enzymes, or bile salts has been displayed. The influence of three VK 2 homologs: VK 2 -MK4, VK 2 -MK7 and VK 2 -MK9 on apoptosis and necrosis of Caco-2 cells was tested proving the lack of their harmful effects on the tested cells. Moreover, the unique role of long-chain homologs (VK 2 -MK9 and VK 2 -MK7) in inhibiting the secretion of pro-inflammatory cytokines such as IL-8 (for Caco-2 tissue) and IL-6 and TNFα (for RAW 264.7) has been documented., (© 2023. The Author(s).)

Preeclampsia (PE) is a prevalent obstetric illness affecting pregnant women worldwide. This comprehensive literature review aims to examine the role of biomarkers and understand the molecular mechanisms underlying PE. The review encompasses studies on biomarkers for predicting, diagnosing, and monitoring PE, focusing on their molecular mechanisms in maternal blood or urine samples. Past research has advanced our understanding of PE pathogenesis, but the etiology remains unclear. Biomarkers such as PlGF, sFlt-1, PP-13, and PAPP-A have shown promise in risk classification and preventive measures, although challenges exist, including low detection rates and discrepancies in predicting different PE subtypes. Future perspectives highlight the importance of larger prospective studies to explore predictive biomarkers and their molecular mechanisms, improving screening efficacy and distinguishing between early-onset and late-onset PE. Biomarker assessments offer reliable and cost-effective screening methods for early detection, prognosis, and monitoring of PE. Early identification of high-risk women enables timely intervention, preventing adverse outcomes. Further research is needed to validate and optimize biomarker models for accurate prediction and diagnosis, ultimately improving maternal and fetal health outcomes.

Objective: Lung cystic fibrosis (CF) is characterized by chronic infections and hyperinflammatory response of neutrophils and macrophages. P. aeruginosa (PA) and S. aureus (MSSA, MRSA) are major pathogens of advanced CF. The main goal of this study was to compare the inflammatory phenotype of murine C57BL/6 macrophages exposed to PA57 with that exposed to MSSA60, both strains isolated from the same patient with severe CF. In the present study, we used C57BL/6 mice sensitive to lung infection with P. aeruginosa., Methods: We measured the release of cytokines and the expression of phenotypic markers of murine neutrophils and macrophages exposed to bacterial cells and biofilm components (i.e., EPS) of the selected bacteria. In addition, a quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages., Results: Neutrophils stimulated with PA57 and MSSA60 strains produced hyperinflammatory pattern of cytokines. The pro-inflammatory impact of PA57 was significantly higher than that of MSSA60 (IL-6/IL-10 ratio: PA57 = 9.3 vs. MSSA60 = 1.7). Macrophages produced significantly lower amount of cytokines, but showed classical pattern of M1 markers (iNOS-High; arginase-1 and mannose receptor MRC1-Low). Importantly, as evidenced by proteomic analysis, PA57 and PA57-EPS were stronger inducers of M1 macrophage polarization than the MSSA60 counterparts., Conclusions: Our study demonstrated that strong biofilm P. aeruginosa strains, CF isolates, are dominant inducers of M1 macrophages, termed biofilm-associated macrophages (BAMs). We suggest that repolarization of detrimental BAMs might be a new therapeutic strategy to ameliorate the airway damage in CF., (© 2023. The Author(s).)

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of β-lactam resistance was determined. Virulence genes, inv A, fim A, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella , no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.

Many difficult-to-treat human infections related to catheters and other indwelling devices are caused by bacteria residing in biofilms. One of the key properties of microorganisms residing in a biofilm is decreased susceptibility towards antimicrobial agents. Therefore, many different approaches have been researched to destroy or inhibit biofilm production by bacteria. Different iminosugars (IS) were reported to inhibit biofilm formation in S. mutans , S. aureus, and P. aeruginosa . The aim of this study was to look for a spectrum of the activity in one of these IS. The iminosugar PDIA beta-1-C-propyl-1,4-dideoxy-1,4-imino-L-arabinitol was tested in vitro at the same concentration against 30 different strains of the most important Gram-negative and Gram-positive human pathogens looking for their biofilm production and viability at different time intervals. It appeared that PDIA inhibited biofilm production of Enterobacter spp., P. aeruginosa , Enterococcus spp. and S. aureus in 8 h, and Klebsiella spp., Acinetobacter spp. and S.epidermidis in 24 h. PDIA caused no growth inhibition of the tested bacteria at a concentration of 0.9 mM. Our results indicate a broad-spectrum biofilm inhibitory activity of PDIA. which may be the basis for future application studies that will help in control of the associated device and biofilm-related infections caused by a wide spectrum of the causative agents.

Background: Campylobacteriosis is currently the most frequently reported zoonosis. Dogs, especially puppies or those with diarrhea, are considered a possible source of human infection. Probiotic bacteria, such as Lactobacillus species, seem to be a valuable tool in controlling of intestinal pathogenic microorganisms in dogs. The main purpose of this study was to assess the anti-Campylobacter activity and some probiotic properties, like ability to produce H 2 O 2, bile salt and low pH tolerance of Lactobacillus strains isolated from gastrointestinal tract of healthy dogs., Results: A total of 39 rectal swabs derived from healthy dogs and 19 from dogs with diarrhea were examined to detect Lactobacillus and Campylobacter bacteria respectively. In total, 30 strains of Lactobacillus genus and four strains of Campylobacter genus were isolated and identified. Of the 30 strains of Lactobacillus, 22 showed an inhibitory effect towards Campylobacter. Four strains with the strongest antagonism towards Campylobacter bacteria (L. salivarius 25 K/L/1, L. rhamnosus 42 K/L/2, L. sakei 50 K/L/1 and L. agilis 55 K/L/1) were selected to assess their potential probiotic traits. Three out of four analyzed strains produced extracellular H 2 O 2. All displayed very good or moderate survival at pH 3.0 and 2.0 and showed high tolerance to 0.5% and 1% bile salts., Conclusions: Among selected Lactobacillus strains, all may have a potential probiotic application in reducing Campylobacter spp. in dogs and thus prevent transmission of infection to humans, although the best candidate for probiotic seems to be L. sakei 50 K/L/1. Further in vitro and in vivo studies are needed., (© 2022. The Author(s).)

Elucidating the mechanisms of bacterial translocation is crucial for the prevention and treatment of neonatal sepsis. In the present study, we aimed to evaluate the potential of lactoferrin to inhibit the development of late-onset blood infection in neonates. Our investigation evaluates the role of key stress factors leading to the translocation of intestinal bacteria into the bloodstream and, consequently, the development of life-threatening sepsis. Three stress factors, namely weaning, intraperitoneal administration of Gram-positive cocci and oral intake of Gram-negative rods, were found to act synergistically. We developed a novel model of rat pups sepsis induced by bacterial translocation and observed the inhibition of this process by supplementation of various forms of lactoferrin: iron-depleted (apolactoferrin), iron-saturated (hololactoferrin) and manganese-saturated lactoferrin. Additionally, lactoferrin saturated with manganese significantly increases the Lactobacillus bacterial population, which contributes to the fortification of the intestinal barrier and inhibits the translocation phenomenon. The acquired knowledge can be used to limit the development of sepsis in newborns in hospital neonatal intensive care units.

Advanced cystic fibrosis (CF) lung disease is commonly characterized by a chronic Pseudomonas aeruginosa infection and destructive inflammation caused by neutrophils. However, the lack of convincing evidence from most informative biomarkers of severe lung dysfunction (SLD-CF) has hampered the formulation of a conclusive, targeted diagnosis of CF. The aim of this study was to determine whether SLD-CF is related to the high concentration of sputum inflammatory mediators and the presence of biofilm-forming bacterial strains. Forty-one patients with advanced CF lung disease were studied. The severity of pulmonary dysfunction was defined by forced expiratory volume in 1 second (FEV1) < 40%. C-reactive protein (CRP) and NLR (neutrophil-lymphocyte ratio) were examined as representative blood-based markers of inflammation. Expectorated sputum was collected and analysed for cytokines and neutrophil-derived defence proteins. Isolated sputum bacteria were identified and their biofilm-forming capacity was determined. There was no association between FEV1% and total number of sputum bacteria. However, in the high biofilm-forming group the median FEV1 was < 40%. Importantly, high density of sputum bacteria was associated with increased concentrations of neutrophil elastase and interleukin (IL)-8 and low concentrations of IL-6 and IL-10. The low concentration of sputum IL-6 is unique for CF and distinct from that observed in other chronic pulmonary inflammatory diseases. These findings strongly suggest that expectorated sputum is an informative source of pulmonary biomarkers representative for advanced CF and may replace more invasive bronchoalveolar lavage analysis to monitor the disease. We recommend to use of the following inflammatory biomarkers: blood CRP, NLR and sputum elastase, IL-6, IL-8 and IL-10., (© 2021 British Society for Immunology.)

Vaginitis in female dogs is a problem most veterinarians face in their practice. It manifests as localized inflammation, and its variable etiology and different severities often make diagnosis problematic. The study consisted of comparing blood smears taken from 16 animals: 8 healthy bitches and 8 bitches with confirmed vaginitis. We analyzed the percentage of different types of white blood cells (leukogram) and changes in the shape of red blood cells (erythrogram) in both groups. We observed changes in red blood cell morphology, i.e., a higher percentage of lacrimocytes and schistocytes in female dogs with vaginitis compared to their healthy counterparts. The observed hematological changes may illustrate the severity of inflammation. The analysis of erythrograms showed a significantly higher percentage of lacrimocytes and schistocytes in diseased bitches (1.58 ± 1.19% and 0.13 ± 0.12%) compared to healthy animals (0.58 ± 0.38 and 0.00 ± 0.00, respectively). The obtained results may indicate that the analysis of erythrograms throughout the course of vaginitis in bitches may constitute a diagnostic tool, as opposed to the analysis of leukograms, which is more sensitive when it comes to the systemic inflammatory response of the organism. It seems that simultaneous analysis of erythrograms and leukograms may facilitate the diagnostic process in clinical practice.