Electrochemical detection of bacterial endotoxin lipopolysaccharide (LPS) on gold electrode modified with DAL-PEG-DK5-PEG-OH - Antimicrobial peptide conjugate.

This work describes fabrication of gold electrodes modified with peptide conjugate DAL-PEG-DK5-PEG-OH that enables ultra-sensitive detection of lipopolysaccharide (LPS) isolated from the reference strain of Escherichia coli O26:B6. The initial step of the established procedure implies immobilization of the fully protected DAL-PEG-DK5-PEG-OH peptide on the surface of the gold electrode previously modified by cysteamine. Then side chain- and Fmoc-deprotection was performed in situ on the electrode surface, followed by its incubation in 1 % of BSA solution to block non-specific bindings sites before LPS detection. The efficiency of the modification was confirmed by X-ray Photoelectron Spectroscopy (XPS) measurements. Additionally, the cyclic voltammetry (CV) and electrochemical impendance spectroscopy (EIS) were employed to monitor the effectiveness of each step of the modification. The obtained results confirmed that the presence of the surface-attached covalently bound peptide DAL-PEG-DK5-PEG-OH enables LPS detection by means of CV technique within the range from 5 × 10-13 to 5 × 10-4 g/mL in PBS solution. The established limit of detection (LOD) for EIS measurements was 4.93 × 10-21 g/mL with wide linear detection range from 5 × 10-21 to 5 × 10-14 g/mL in PBS solution. Furthermore, we confirmed the ability of the electrode to detect LPS in a complex biological samples, like mouse urine and human serum. The effectiveness of the electrodes in identifying LPS in both urine and serum matrices was confirmed for samples containing LPS at both 2.5 × 10-15 g/mL and 2.5 × 10-9 g/mL.

Depletion of Mcpip1 in murine myeloid cells results in intestinal dysbiosis followed by allergic inflammation.

MCPIP1 (called also Regnase-1) is a negative regulator of inflammation. Knockout of the Zc3h12a gene, encoding Mcpip1 in cells of myeloid origin (Mcpip1MKO), has a pathological effect on many organs. The aim of this study was to comprehensively analyze pathological changes in the skin caused by Mcpip1 deficiency in phagocytes with an emphasis on its molecular mechanism associated with microbiome dysbiosis. Mcpip1MKO mice exhibited spontaneous wound formation on the skin. On a molecular level, the Th2-type immune response was predominantly characterized by an increase in Il5 and Il13 transcript levels, as well as eosinophil and mast cell infiltration. Irritation by DNFB led to a more severe skin contact allergy in Mcpip1MKO mice. Allergic reactions on the skin were strongly influenced by gut dysbiosis and enhanced systemic dissemination of bacteria. This process was followed by activation of the C/EBP pathway in peripheral macrophages, leading to local changes in the cytokine microenvironment that promoted the Th2 response. A reduced bacterial load inhibited allergic inflammation, indicating the role of intestinal dysbiosis in the development of skin diseases. Our results clearly show that MCPIP1 in phagocytes is an essential negative regulator that controls the gut-skin axis.

Studies of Streptococcus anginosus Virulence in Dictyostelium discoideum and Galleria mellonella Models.

For many years, Streptococcus anginosus has been considered a commensal colonizing the oral cavity, as well as the gastrointestinal and genitourinary tracts. However, recent epidemiological and clinical data designate this bacterium as an emerging opportunistic pathogen. Despite the reported pathogenicity of S. anginosus, the molecular mechanism underpinning its virulence is poorly described. Therefore, our goal was to develop and optimize efficient and simple infection models that can be applied to examine the virulence of S. anginosus and to study host-pathogen interactions. Using 23 S. anginosus isolates collected from different infections, including severe and superficial infections, as well as an attenuated strain devoid of CppA, we demonstrate for the first time that Dictyostelium discoideum is a suitable model for initial, fast, and large-scale screening of virulence. Furthermore, we found that another nonvertebrate animal model, Galleria mellonella, can be used to study the pathogenesis of S. anginosus infection, with an emphasis on the interactions between the pathogen and host innate immunity. Examining the profile of immune defense genes, including antimicrobial peptides, opsonins, regulators of nodulation, and inhibitors of proteases, by quantitative PCR (qPCR) we identified different immune response profiles depending on the S. anginosus strain. Using these models, we show that S. anginosus is resistant to the bactericidal activity of phagocytes, a phenomenon confirmed using human neutrophils. Notably, since we found that the data from these models corresponded to the clinical severity of infection, we propose their further application to studies of the virulence of S. anginosus.

The Clinical View on Streptococcus anginosus Group - Opportunistic Pathogens Coming Out of Hiding.

Three distinct streptococcal species: Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus, belonging to the Streptococcus anginosus group (SAG), also known as Streptococcus milleri group, have been attracting clinicians and microbiologists, not only as oral commensals but also as opportunistic pathogens. For years they have been simply classified as so called viridans streptococci, and distinct species were not associated with particular clinical manifestations. Therefore, description of SAG members are clearly underrepresented in the literature, compared to other medically relevant streptococci. However, the increasing number of reports of life-threatening infections caused by SAG indicates their emerging pathogenicity. The improved clinical data generated with the application of modern molecular diagnostic techniques allow for precise identification of individual species belonging to SAG. This review summarizes clinical reports on SAG infections and systematizes data on the occurrence of individual species at the site of infection. We also discuss the issue of proper microbiological diagnostics, which is crucial for further clinical treatment.

The Multi-Component Causes of Late Neonatal Sepsis-Can We Regulate Them?

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.

Lactoferrin Metal Saturation-Which Form Is the Best for Neonatal Nutrition?

We evaluated the impact of metal saturation of lactoferrin (with iron and manganese) on population numbers of pathogenic species relevant for neonatal sepsis that commonly originates from the gut due to bacterial translocation. Little attention has been paid to how metal ions bound to the protein affect its activity. Several reference and clinical strains as well as probiotic strains were incubated with different forms of lactoferrin: metal-depleted (apolactoferrin), iron-saturated (hololactoferrin) and manganese-saturated lactoferrin. We also attempted to confirm the observed effects of lactoferrin forms in vivo using rat pups. The observed decrease in population numbers of Gram-negative rods could not be confirmed by quantitative plating-lactoferrin may regulate these populations diversely (e.g., by anti-biofilm activity) and contribute to the inhibition of inflammatory response. We did not see any effect of lactoferrin forms on staphylococci and bifidobacteria. However, we have noted a significant increase of population numbers of Lactobacillus strains upon incubation with manganese-saturated lactoferrin. These results were confirmed in vivo in a rat model. Metal saturation is an underestimated factor regulating lactoferrin activity. Some forms are more potent in the inhibition of pathogenic species while others, such as manganese-saturated lactoferrin, could contribute to the restoration of gut homeostasis.

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.

Molecular Epidemiology and Drug Resistance of Acinetobacter baumannii Isolated from Hospitals in Southern Poland: ICU as a Risk Factor for XDR Strains.

The objectives of the present study were to investigate the carbapenemase and metallo-beta-lactamase genes of Acinetobacter baumannii clinical isolates by polymerase chain reaction (PCR) and real time PCR and to determine the molecular epidemiology of the strains using the DiversiLab tool. From these data, correlations between drug resistance, resistance genes, and epidemiological clones may be revealed. The study was conducted on 125 A. baumannii collected over the 2013 year. The majority of the isolates from both intensive care unit (ICU) and non-ICU cases originated from pneumonia infections (79.2%), isolates from blood infections accounted for 17.6% and 3.2% were from meningitis infections. In the ICU cases compared with the non-ICU cases, bloodstream infections were more frequently diagnosed (19.2% vs. 11.5%). Sixty percent of A. baumannii strains were resistant to all the antimicrobials tested with the exception of colistin. All strains were susceptible to colistin and polymyxin B. Extensively drug-resistant (XDR) strains accounted for 80.8% of the isolates tested and these XDR strains were more frequently isolated from ICU cases than from non-ICU cases (93.9% vs. 30.8%). Among the 101 isolates of A. baumannii exhibiting the XDR pattern of resistance, 80 possessed the blaOXA-24 gene and 29 had the blaOXA-23 gene. Only two isolates possessed the blaVIM gene. The presence of the ISAba1element was confirmed among 10 strains from patients hospitalized in the ICU. Using repetitive extragenic palindromic sequence PCR (DiversiLab typing), six clones and 12 unique strains were identified, of which two clones dominated. Most isolates belonging to clone 1 (66.7%) and clone 2 (85.5%) were susceptible only to colistin. In summary, it is clear from our findings and those of other studies that carbapenem resistance among A. baumannii strains presents a serious clinical problem worldwide. Furthermore, the presence of XDR international clone II in ICUs poses a potential risk for future outbreaks of A. baumannii infection and controlling A. baumannii infections in hospitals presents a serious challenge.

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.

Bacterial infections of the lower genital tract in fertile and infertile women from the southeastern Poland.

OBJECTIVES: The objective of the study was to investigate the detection rates of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Gardnerella vaginalis, Escherichia coli, Streptococcus agalactiae and Enterococcus faecalis, showing no clinical signs of an ongoing, acute inflammatory state of the vagina and/or the cenrvix, in fertile and infertile women. MATERIAL AND METHODS: The study encompassed 161 women, including 101 women treated for infertility and 60 fertile women who had already given birth to healthy children. The material for the presence of C. trachomatis, N. gonorrhoeae, M. genitalium, M. hominis and U. urealyticum was collected from the cervical canal and analyzed by PCR. Furthermore, BD ProbeTec ET system was used to detect C. trachomatis infection. Vaginal swabs were collected for classification of bacterial vaginosis and aerobic vaginitis and assessed according to the Nugent score, as well as by traditional culture methods. RESULTS: U. urealyticum was identified in 9% of the infertile women and in 8% of controls. Presence of M. hominis was demonstrated only in the former (4%) and C. trachomatis only in latter (3%). N. gonorrhoeae and M. genitalium were not found in any of the examined women. The frequency of aerobic vaginitis in both groups was estimated at 12%. There were 7% bacterial vaginosis cases in the study group, and none in the control group (p=0.0096). CONCLUSIONS: Despite having no symptoms of an ongoing acute inflammation of the reproductive tract, many women may experience permanent or periodic shifts of equilibrium of the vaginal and/or cervical microflora. BV develops more frequently in infertile patients when compared to the fertile women.

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.