Saccharomyces cerevisiae ß-glucan improves the response of trained macrophages to severe P. aeruginosa infections.

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 ß-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 ß-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 ß-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 ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.

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.

Biofilm-forming strains of P. aeruginosa and S. aureus isolated from cystic fibrosis patients differently affect inflammatory phenotype of macrophages.

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.

In Vitro assessment of anti-Campylobacter activity of lactobacillus strains isolated from canine rectal swabs.

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 H2O2, 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 H2O2. 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.

Chronic bacterial pulmonary infections in advanced cystic fibrosis differently affect the level of sputum neutrophil elastase, IL-8 and IL-6.

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.

The vaginal microflora changes in various stages of the estrous cycle of healthy female dogs and the ones with genital tract infections.

BACKGROUND: Inflammatory diseases of reproductive tract in bitches are a common problem in veterinary practice. The inflammation can lead to serious health problems. Research to determine the correlation between the health status of females, phase of the cycle, age and bacterial flora of the genital tract has been ongoing for years, but the results obtained by individual authors are often contradictory. RESULTS: A total of 39 dogs were included in this study. Ten were qualified to the 1st group with genital tract infections (8 in anestrus and 2 in proestrus) and 29 to the 2nd group without such infections (16 in anestrus, 9 in proestrus and 4 in diestrus). The most common bacterial isolates obtained from the vaginal tract of all dogs were Escherichia coli, Staphylococcus pseudintermedius and Streptococcus canis. The prevalence of Gram-negative rods (other than E. coli) was significantly higher in the group with genital tract infections versus healthy dogs. There was no presence of Chlamydiaceae, Chlamydia abortus and lactic acid-producing bacteria in tested swabs. CONCLUSIONS: Our study identified the most common bacteria in the genital tract of bitches. The total number of bacteria was almost the same in the healthy and infected dogs, as well as between the cycle stages. In our opinion, bacterial culturing of vaginal swab specimens from bitches without signs of genital disease is of little value. Furthermore, it should always be preceded by clinical examination and cytological examination of the vaginal epithelium.

Pseudomonas aeruginosa biofilm is a potent inducer of phagocyte hyperinflammation.

OBJECTIVE: Pseudomonas aeruginosa effectively facilitate resistance to phagocyte killing by biofilm formation. However, the cross talk between biofilm components and phagocytes is still unclear. We hypothesize that a biofilm provides a concentrated extracellular source of LPS, DNA and exopolysaccharides (EPS), which polarize neighbouring phagocytes into an adverse hyperinflammatory state of activation. METHODS: We measured the release of a panel of mediators produced in vitro by murine neutrophils and macrophages exposed to various biofilm components of P. aeruginosa cultures. RESULTS: We found that conditioned media from a high biofilm-producing strain of P. aeruginosa, PAR5, accumulated high concentrations of extracellular bacterial LPS, DNA and EPS by 72 h. These conditioned media induced phagocytes to release a hyperinflammatory pattern of mediators, with enhanced levels of TNF-α, IL-6, IL12p40, PGE2 and NO. Moreover, the phagocytes also upregulated COX-2 and iNOS with no influence on the expression of arginase-1. CONCLUSIONS: Phagocytes exposed to biofilm microenvironment, called by us biofilm-associated neutrophils/macrophages (BANs/BAMs), display secretory properties similar to that of N1/M1-type phagocytes. These results suggest that in vivo high concentrations of LPS and DNA, trapped in biofilm by EPS, might convert infiltrating phagocytes into cells responsible for tissue injury without direct contact with bacteria and phagocytosis.

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.

Genital Chlamydia trachomatis infections in young adults - a school-based bio-behavioural study in urban areas, Poland, 2012 to 2015.

Context and AimOne of the most common sexually transmitted pathogens disproportionately affecting young people is Chlamydia trachomatis (CT). This study aimed to assess prevalence of CT among sexually active students (aged 18-19 years) in their final years of high school education in Warsaw and Krakow. Methods: The sample was selected from 61 clusters, each cluster representing one school. We described city, sex, type of school and their association with CT prevalence. To account for non-responders we applied inverse probability weighting. Results: Our study population consisted of 3,136 young adults eligible for CT screening, of whom 2,326 reported having had sexual intercourse within past 12 months. Of the 950 students who agreed to be tested, 39 were infected with CT. Weighted prevalence of CT was 3.9% (95% confidence interval (CI): 2.7-5.1); however, prevalence in the students in Warsaw (6.6%; 95% CI: 3.5-12.4) was six times higher (prevalence ratio (PR) = 5.9; 95% CI: 2.0-17.3) than in Krakow (1.1%; 95% CI: 0.5-2.6). In both settings, female students attending vocational-technical schools were most affected; the prevalence in this group was more than five times higher (PR = 5.2; 95% CI: 1.7-15.6) compared with female peers in high schools and more than three times higher (PR = 3.3; 95% CI: 1.0-10.7) compared with male peers attending vocational-technical schools. Conclusion: Our study suggested prevalence of CT infection among young people in Poland comparable with the European average, supporting implementation of a CT control programme as recommended in international guidelines.

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 dual role of Escherichia coli in the course of ulcerative colitis.

BACKGROUND: This study examines the dual role of Escherichia coli in the course of ulcerative colitis (UC). The intestinal microbiota is considered to play an important role in UC pathogenesis, but how E. coli contributes to inflammation in UC is still unknown. On the one hand, we demonstrated that there was a significant increase in the number of E. coli at the sites of inflammation in patients with UC, which can lead to immune system activation, whilst, on the other hand, E. coli may contribute to the resolution of inflammatory reactions since E. coli can inhibit hydroxyl radical formation by eliminating substrates of the Fenton reaction, by assimilating ferrous iron (Fe2+) and inducing the decomposition of hydrogen peroxide (H2O2). On this way, E. coli may affect the initiation and/or prolongation of remission stages of UC. METHODS: Ten E. coli strains were isolated from the colonic mucosa of patients in the acute phase of UC. Using PCR, we examined the presence of genes encoding catalases (katG and katE) and proteins participating in iron acquisition (feoB, fepA, fhuA, fecA, iroN, fyuA, and iutA) in these E. coli strains. To determine if iron ions influence the growth rate of E. coli and its ability to decompose H2O2, we grew E. coli in defined culture media without iron (M9(-)) or with ferrous ions (M9(Fe2+)). Expression levels of genes encoding catalases were examined by real-time PCR. RESULTS: All investigated E. coli strains had catalase genes (katG, katE), genes coding for receptors for Fe2+ (feoB) and at least one of the genes responsible for iron acquisition related to siderophores (fepA, fhuA, fecA, iroN, fyuA, iutA). E. coli cultured in M9(Fe2+) grew faster than E. coli in M9(-). The presence of Fe2+ in the media contributed to the increased rate of H2O2 decomposition by E. coli and induced katG gene expression. CONCLUSIONS: E. coli eliminates substrates of the Fenton reaction by assimilating Fe2+ and biosynthesizing enzymes that catalyze H2O2 decomposition. Thus, E. coli can inhibit hydroxyl radical formation, and affects the initiation and/or prolongation of remission stages of UC.

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.

Staphylococcus epidermidis and biofilm-associated neutrophils in chronic rhinosinusitis. A pilot study.

A key role of bacterial biofilm in the pathogenesis of chronic rhinosinusitis (CRS) with (CRSwNP) and without nasal polyps (CRSsNP) is commonly accepted. However, the impact of some bacterial species isolated from inflamed sinus mucosa on biofilm formation is unclear. In particular, the role of Staphylococcus epidermidis as aetiological agents of CRS is controversial. Moreover, the effect of biofilm formation on neutrophil infiltration and activity in CRSwNP calls for explanation. In this study, biofilms were found in three of 10 patients (mean age = 46 ± 14) with CRS undergoing endoscopic sinus surgery by means of scanning electron microscopy. Unexpectedly, S. epidermidis was the primary isolated bacteria and was also found to be present in all biofilm-positive mucosa specimens, indicating its pivotal role in the pathogenesis of severe chronic infections associated with biofilm formation. We have also measured the activity of myeloperoxidase (MPO), the most abundant neutrophil enzyme, to demonstrate the presence of neutrophils in the samples tested. Our present results show that the level of MPO in CRS associated with biofilm is lower than that without biofilm. It may suggest either a low number of neutrophils or the presence of a type of neutrophils with compromised antimicrobial activity, described as biofilm-associated neutrophils (BAN). Finally, we conclude that further studies with a large number of CRS cases should be performed to establish the association between S. epidermidis and other frequently isolated bacterial species from paranasal sinuses, with the severity of CRS, biofilm formation and the infiltration of BAN.

Visible light induced photocatalytic inactivation of bacteria by modified titanium dioxide films on organic polymers.

Commercially available polypropylene foil was pretreated with a low temperature oxygen plasma and covered with a thin film of nanocrystalline titanium dioxide by dip coating. The films were then photosensitized by titanium(IV) surface charge transfer complexes formed by impregnation with catechol. The photoactivity of the coatings up to 460 nm was confirmed by photoelectrochemical measurements. The photoinactivation of Escherichia coli and Staphylococcus aureus was evaluated by a glass adhesion test based on ISO 27447:2009(E) in the presence of visible light. The coating showed good antimicrobial activity induced by light from a light-emitting diode (405 nm), in particular towards E. coli ATCC 25922 strain. Adaptation of ISO 27447:2009(E) to assess bacterial photoinactivation by photocatalytic coatings will allow this procedure to be applied for the comparison of photoactivity under a range of irradiation conditions.

Supplementation of standard antibiotic therapy with oral probiotics for bacterial vaginosis and aerobic vaginitis: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: This multicentre, randomised, double-blind, placebo-controlled trial was performed to determine whether the use of oral probiotic preparation (prOVag®) containing three Lactobacillus strains together with standard metronidazole treatment and also targeted antibiotic treatment (following the failure of metronidazole therapy) could reduce the recurrence rates of bacterial vaginosis (BV) and aerobic vaginitis (AV). METHODS: Patients at private gynaecological clinics in Poland with histories of recurrent BV/AV and current symptoms were randomly allocated to receive metronidazole and probiotic or placebo, and assessed monthly on visits II and III-V. The total number of study visits was 5-6 (I, II, II bis - if applicable, III, IV, V). One probiotic or placebo capsule was administered with metronidazole/targeted antibiotic twice daily for 10 days; during follow up, patients took one capsule daily for 10 days perimenstrually. Clinical examination and vaginal swabbing were performed at each visit. Primary outcomes were clinical or microbiological BV/AV recurrence and probiotic safety. Secondary outcomes were vaginal pH, Nugent score, and Lactobacillus counts in the vaginal microbiota. Safety analysis was performed in 578 (probiotic, n = 285; placebo, n = 293) 18-50-year-old women who were randomised. RESULTS: BV/AV was confirmed microbiologically in 241 (probiotic, n = 118; placebo, n = 123) participants, who continued the trial. Data from 154 (probiotic, n = 73; placebo, n = 81) participants who completed the study were analysed to determine the efficacy of prOVag. Additional analyses included 37 (probiotic, n = 22; placebo, n = 15) participants who received targeted antibiotics and probiotics or placebo. prOVag lengthened the time to clinical relapse of BV/AV symptoms up to 51 % (p < 0.05) compared with placebo; AV relapse was delayed by up to 76 % (p < 0.05). Probiotic use also reduced and maintained low vaginal pH and Nugent score, and increased vaginal Lactobacillus counts following standard treatment. CONCLUSION: This study demonstrated that oral probiotics lengthened remission in patients with recurrent BV/AV and improved clinical and microbiological parameters. TRIAL REGISTRATION: NCT01993524 ; 20 November 2013.

Distinct effects of Lactobacillus plantarum KL30B and Escherichia coli 3A1 on the induction and development of acute and chronic inflammation.

OBJECTIVE: Enteric bacteria are involved in the pathogenesis of ulcerative colitis. In experimental colitis, a breakdown of the intestinal epithelial barrier results in inflow of various gut bacteria, induction of acute inflammation and finally, progression to chronic colitis. MATERIAL AND METHODS: In the present study we compared pro-inflammatory properties of two bacterial strains isolated from human microbiome, Escherichia coli 3A1 and Lactobacillus plantarum KL30B. The study was performed using two experimental models of acute inflammation: peritonitis in mice and trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. RESULTS: Both bacterial strains induced massive neutrophil infiltration upon injection into sterile peritoneal cavity. However, peritoneal exudate cells stimulated in vitro with E. coli 3A1, produced far more nitric oxide, than those stimulated with L. plantarum KL30B. Interestingly, distinct effect on the development of TNBS-induced colitis was observed after oral administration of the tested bacteria. Lactobacillus plantarum KL30B evoked strong acute colitis. On the contrary, the administration of E. coli 3A1 resulted in a progression of colitis to chronicity. CONCLUSIONS: Our results show that distinct effects of bacterial administration on the development of ongoing inflammation is strain specific and depends on the final effect of cross-talk between bacteria and cells of the innate immune system.

Possible role of Escherichia coli in propagation and perpetuation of chronic inflammation in ulcerative colitis.

BACKGROUND: This study investigated a possible role of Escherichia coli in propagation and perpetuation of the chronic inflammation in ulcerative colitis (UC). The lesions of UC are located superficially on the rectal and/or colonic mucosa. It is suggested that the commensal bacteria of the digestive tract may play a role in the pathogenesis of UC. Several studies have demonstrated proliferation of E. coli in the gut of UC patients. An increase in the number of E. coli in the inflamed tissue is most probably related to the abundance of iron ions produced by the bacteria. METHODS: Colon mucosal biopsies were collected from 30 patients with acute-phase UC, both from tissues with inflammatory changes (n = 30) and unchanged tissue with no inflammatory changes (n = 30) from the same patient. Biopsies were also taken from 16 patients with irritable bowel syndrome diarrhea who comprised the control group. Quantitative and qualitative analysis of the biopsy specimens was performed using culture methods and real-time polymerase chain reaction (PCR). Genotyping of the E. coli isolates was done using pulsed-field gel electrophoresis. Multiplex PCR was used to compare the E. coli strains for the presence of genes responsible for synthesis of iron acquisition proteins: iroN, iutA, iha, ireA, chuA, and hlyA. RESULTS: We demonstrated that there was a significant increase in the number of E. coli at the sites of inflammation in patients with UC compared to the control group (P = 0.031). Comparative analysis of the restriction patterns of E. coli isolated from inflammatory and unchanged tissues showed that the local inflammatory changes did not promote specific E. coli strains. There was a significant difference in the frequency of the iroN gene in E. coli isolated from patients with UC as compared to the control group. CONCLUSIONS: The increase in the numbers of E. coli in the inflammatory tissues is related to the presence of chuA and iutA genes, which facilitate iron acquisition during chronic intestinal inflammatory processes.

A high-throughput method for the quantification of iron saturation in lactoferrin preparations.

Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A(280/466)). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.

Influence of taurine haloamines (TauCl and TauBr) on the development of Pseudomonas aeruginosa biofilm: a preliminary study.

Biofilms are consortia of microorganisms (sessile cells) that form on various surfaces including mucosal membranes or teeth. Bacterial biofilms cause many human infections such as chronic sinusitis, acne vulgaris, periodontal diseases, and chronic wounds. These infections are persistent as they show increased resistance to antibiotics and host defense system. Taurine chloramine (TauCl) and taurine bromamine (TauBr) are the physiological products of activated neutrophils, resulting from the reaction between taurine with hypochlorous acid (HOCl) and hypobromous acid (HOBr), respectively. It has been shown in vitro that taurine haloamines exert antimicrobial properties against various pathogenic bacteria. Moreover, clinical studies have shown that both haloamines are effective in the local treatment of skin and mucose infections, including biofilm-related infections. Nevertheless, it has been not tested yet whether they can kill bacteria hidden in biofilm or disrupt biofilm structure. In this study we have investigated the capacity of TauCl and TauBr to inhibit in vitro the formation of P. aeruginosa biofilm. We have also tested their ability to destroy the mature biofilm. Our results suggest that TauBr is able to inhibit in vitro the formation of P. aeruginosa biofilm but cannot destroy the mature biofilm and effectively killed hidden bacteria. In further studies, the combined effect of TauBr and DNase, one of suggested biofilm inhibitors, was tested. Together, we conclude that TauBr is a better than TauCl candidate for local therapy of biofilm-related infections. However, a combined therapy, an application of TauBr together with other anti-biofilm agents (e.g., DNase), seems to be more promising.

Adherence of group B streptococci to human rectal and vaginal epithelial cell lines in relation to capsular polysaccharides as well as alpha-like protein genes - pilot study.

Streptococcus agalactiae (Group B Streptococci, GBS) constitutes a risk factor for infections of the newborns born by colonized mothers. The adherence of GBS to epithelial cells has been proved to be an important factor in the colonization of mucus membranes of both human rectum and vagina. The objective of the study was to assess the adhesion of the selected GBS strains to the human colon adenocarcinoma cell line (HT-29) and human epidermoid vulvo-vaginal cells (A-431) in relation to the capsular polysaccharides and alpha-like protein genes. GBS strains from the human sources belonging to Ia, Ib, II, III and V serotypes possessing different surface alpha-like protein genes such as the alp 2, alp 3, bca, epsilon and rib in the conventional adherence assay were examined. The adherence of GBS strains to the HT-29 cell line was considerably higher than to the A-431 cell line. For GBS serotype Ia and III, a significant difference between the adhesion to the HT-29 and A-431 cell lines was presented. The adhesion of GBS strains to the HT-29 cell line depended on alpha-like protein genes. The most adhesive ones were the GBS strains containing the rib and alp 2 genes. The adherence of GBS strains to the A-431 cell line depended on both their serotype and alpha-like protein genes. Serotype III adhered to the A-431 cells most tightly, particularly the strains containing the rib and alp 2 genes. GBS strains containing the rib gene adhered to the HT-29 and A-431 cell lines more firmly than GBS strains containing other alpha-like protein genes.