The impact of gut bacteria producing long chain homologs of vitamin K2 on colorectal carcinogenesis.

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 K2 (VK2) 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. VK2 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 K2 synthesized by the most active members of the human gut microbiome. This analysis tested 86 intestinally derived bacterial strains, among which the largest VK2 producers (Lactobacillus, Bifidobacterium, Bacillus) were selected. Moreover, based on the chosen VK2-MK4 homolog, the potential of VK2 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 VK2 homologs: VK2-MK4, VK2-MK7 and VK2-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 (VK2-MK9 and VK2-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.

Coagulase-Negative Staphylococci Contained in Gut Microbiota as a Primary Source of Sepsis in Low- and Very Low Birth Weight Neonates.

BACKGROUND: There are only a few reports in the literature about translocation of coagulase-negative staphylococci (CoNS) as a primary cause of sepsis in neonates, although CoNS are among a short list of "translocating" bacteria when present in abundance. METHODS: 468 blood samples, 119 stool samples, and 8 catheter tips, from 311 neonates, were tested for presence of microorganisms. CoNS strains isolated from the blood and stool or from blood and catheter tip of the same newborn at approximately the same time were paired and typed with PFGE (Pulse-Field Gel Electrophoresis) method. The strains were then tested for the presence of adherence genes and biofilm formation. RESULTS: The strains with identical PFGE profiles in comparison to those with non-identical profiles differed in terms of the pattern of the virulence genes and showed a lack of the genes related to adherence, but more often presence of IS256, which is related to virulence. They also were phenotypically unable to adhere to intestinal Caco2 cells. CONCLUSIONS: A considerable proportion of CoNS strains isolated from bloodstream of VLBW/LWB neonates was identical to the strains isolated from faeces of the same neonates at the same time. These observations may offer indirect evidence indicating that at least some CoNS can translocate from the gastrointestinal tract of the premature neonates into the bloodstream and thus cause generalized infection.

Analysis of proteinuria in experimental model of ascending acute kidney injury.

Proteinuria accompanies kidney diseases of various etiology and correlates with the degree of organ damage. Analysis of proteinuria allows the location of pathophysiological process in the kidney, and assessment of the severity of the kidney disease in chronic and acute kidney injury (AKI). Ascending bacterial acute kidney injury develops as a consequence of pyelonephritis. It is a rare complication in patients with anatomical or functional dysfunctions of the urinary tract. AIM: The aim of the study was to perform the laboratory analysis of proteinuria in bacterial ascending AKI in an experimental model. MATERIALS AND METHODS: Female Wistar rats (n = 24) were intravesically administrated bacterial suspension of Escherichia coli (E. coli) to induce: pyelonephritis (group 1, 105 CFU/ml); AKI (group 2, 107 CFU/ml); AKI and urosepsis (group 3, 109 CFU/ml) respectively. Bacterial strain - E.coli, was isolated from a patient with acute pyelonephritis. The daily diuresis and urine protein excretion was measured the following days: 0, 7, 14 and 21. Moreover, electrophoretic separation of urine protein, densitometric analysis of albumin fraction and uromodulin concentration in urine were performed. Moreover, the key parameters for the diagnosis of AKI were assayed. RESULTS: Increased urinary protein excretion was observed in each of the study groups. Moreover, the study groups showed significant changes in protein selectivity in the urine. CONCLUSIONS: Moderately severe proteinuria was revealed while its selectivity suggested significant damage of glomeruli and renal tubules in groups with complications caused by AKI induced by ascending pyelonephritis.

Experimental model for acute kidney injury caused by uropathogenic Escherichia coli.

INTRODUCTION: Acute kidney injury (AKI) is the rapid deterioration of renal function, diagnosed on the basis of an increase in serum creatinine and abnormal urinary parameters. AKI is associated with increased risk of mortality or chronic kidney disease (CKD). The aim of the study was to develop an experimental model for AKI resulting from Escherichia coli-induced pyelonephritis. E. coli was isolated from a patient with clinical symptoms of urinary tract infection (UTI). MATERIAL/METHODS: The study included three groups of female Wistar rats (groups 1, 2 and 3), in which pyelonephritis was induced by transurethral inoculation with highly virulent E. coli (105, 107 and 109 cfu/ml, respectively). Urine and blood samples for analysis were obtained prior to the inoculation (day 0), as well as 7, 14 and 21 days thereafter. RESULTS: Aside from a microbiological examination of urine samples, daily urine output, serum creatinine (CreaS), creatinine clearance (CrCl), interleukin 6 (IL-6), fractional excretion of sodium (FENa) and fractional excretion of urea (FEUrea) were determined. A histopathological examination of kidney and urinary bladder specimens was conducted as well. While UTI-related pyelonephritis developed irrespective of E. coli inoculum size, AKI was observed only following transurethral administration of E. coli at the intermediate and high dose, i.e. 107 and 109 cfu/ml, respectively (group 2 and 3). DISCUSSION: An increase in CreaS and abnormal diuresis were accompanied by changes in parameters specific for various forms of AKI, i.e. FENa and FEUrea. Based on these changes, administration of E. coli at 107 cfu/ml was demonstrated to induce renal AKI, whereas inoculation with 109 cfu/ml seemed to cause not only ascending pyelonephritis, but perhaps also bacteremia and urosepsis (prerenal component of AKI).

The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation.

Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.

Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease.

AIM: To determine the features of Enterococcus that contribute to the development and maintenance of the inflammatory process in patients with inflammatory bowel disease (IBD). METHODS: Multiplex polymerase chain reaction (PCR) was applied to assess the presence of genes that encode virulence factors [surface aggregating protein (asa1), gelatinase (gelE), cytolysin (cylA), extracellular surface protein (esp) and hyaluronidase (hyl)] in the genomic DNA of 28 strains of Enterococcus isolated from the intestinal tissues of children with IBD (n = 16) and of children without IBD (controls; n = 12). Additionally, strains with confirmed presence of the gelE gene were tested by PCR for the presence of quorum sensing genes (fsrA, fsrB, fsrC) that control the gelatinase production. Gelatinase activity was tested on agar plates containing 1.6% gelatin. We also analysed the ability of Enterococcus strains to release and decompose hydrogen peroxide (using Analytical Merckoquant peroxide test strips) and tested their ability to adhere to Caco-2 human gut epithelium cells and form biofilms in vitro. RESULTS: A comparison of the genomes of Enterococcus strains isolated from the inflamed mucosa of patients with IBD with those of the control group showed statistically significant differences in the frequency of the asa1 gene and the gelE gene. Furthermore, the cumulative occurrence of different virulence genes in the genome of a single strain of Enterococcus isolated from the IBD patient group is greater than in a strain from the control group, although no significant difference was found. Statistically significant differences in the decomposition of hydrogen peroxide and adherence to the Caco-2 epithelial cell line between the strains from the patient group and control group were demonstrated. The results also showed that profuse biofilm production was more frequent among Enterococcus strains isolated from children with IBD than in control strains. CONCLUSION: Enterococcus strains that adhere strongly to the intestinal epithelium, form biofilms and possess antioxidant defence mechanisms seem to have the greatest influence on the inflammatory process.

Laboratory diagnostics of renal function in an experimental model of ascending pyelonephritis with high-virulent Escherichia coli.

INTRODUCTION: Urinary tract infections (UTI) are caused in 95% of cases by bacteria--E. coli. UTIs usually are limited to the lower urinary tract, but it may also evolve into pyelonephritis and acute kidney injury. OBJECTIVES: The aim of this study was the laboratory evaluation of renal function in an experimental model of ascending pyelonephritis caused by intravesical infusion of E. coli. MATERIAL & METHODS: In female Wistar rats UTI was induced by intravesical administration of E. coli suspension in a dose 10(5) c.f.u./ml (Group 1), and 10(7) c.f.u./ml (Group 2). On the 0,7th, 14th and 21st day of the experiment the animals underwent the procedures of collecting blood and urine samples. RESULTS: The results shown that in group 2 on the 7th and 14th day of the study the creatinine clearance decreased by 36%, and on 21th by 34%. The increase in serum uric acid concentration (micromol/l) in group 2 was observed on the 7th (229.75 +/- 79.05) and 21st day (98.5 +/- 11.33) with respect to day 0 (77.12 +/- 11.63). In group 2 on the 7th day of the experiment there was observed the increased levels of potassium (mmol/l) in serum (13.5 +/- 1.48) with respect to day 0 (7.74 +/- 0.88). In group 2 in the 7th (1.06 +/- 0.18) and 14th day (1.32 +/- 0.26) there was noted the decreased excretion of potassium in the urine (mmol/24h) with respect to day 0 (3.75 +/- 1.9). The decrease in serum sodium levels (mmol/l) in group 2 was recorded on 14th day (121.5 +/- 8.7) with respect to day 0 (131.62 +/- 4.07). Increased factional sodium excretion--FENa (%) was observed in group 2 on 14th day (0.25 +/- 0.06) with respect to day 0 (0.12 +/- 0.06). CONCLUSIONS: Our main finding is that--independently of the amount bacteria present in urinary bladder--in this inflammatory model there occurs inevitably acute kidney injury, however higher bacteria amount depicts a very clear profile of laboratory parameters that point at the kidney impaired function.