Phage Therapy: a Step Forward in the Treatment of Pseudomonas aeruginosa Infections

Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and nowadays, a common infection can be fatal. To address this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since Pseudomonas aeruginosa is one of the most common causes of health care-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review further addresses the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.     

Zur Kenntnis der Wirkung von Gallensalzen auf die Zelle

Zusammenfassung Schon sehr kleine Mengen von Gallensalzen, wie desoxycholsaures und apocholsaures Natrium lassen aus Zellen Stoffe austreten, ohne da\ die Zellen tot sind. Die ausgetretenen Stoffe können mit der Schüttelprobe (Oberflächenspannung), mit der Ninhydrinprobe (Eiwei\bruchstücke), und mit der Silbernitratprobe erfa\t werden.Auch kleine Mengen von Quecksilberchlorid veranlassen Austritt von Stoffen aus der Zelle. Dabei sind die Zellen nicht tot. Bei den Gallensalzproben ist eine untere Grenze der Wirksamkeit schwer anzugeben. Wenn nicht gerade (bei Kartoffelversuchen) Randstücke oder Siebteile mit viel Eiwei\ vorliegen, ist eine Normalgrenze der Wirkung etwa bei 1 75000 bis 1 100000 erreicht, soweit die Ninhydrinprobe in Frage kommt. Bei Gegenwart eiwei\reicher Kartoffelzellen bzw. Siebteile ist die Grenze der mit Ninhydrin zu erfassenden Wirkung viel tiefer anzusetzen, etwa 1 1000000. Durch die physiologischen oder anatomisch-topographischen Sondereigenschaften (viel Eiwei\, viel Siebteile in einzelnen Proben) erhält die Gallensalzmethode bis zu einem gewissen Grad etwas Subjektives. Trotzdem steht unter Umständen der Gallensalzeinflu\ 1 1000000 au\er jedem Zweifel! Die Methodik setzt eben eine gewisse Erfahrung voraus.Bei höheren Pflanzen kann mit der Neutralsalz-Gallensalzbehandlung der Unterschied zwischen Epidermiszellen und Spaltöffnungsapparat ungemein scharf vordemonstriert werden. Bei geeigneter Behandlung lösen sich Zellkerne schrittweise in Neutralsalz-Gallensalz auf, die Kerne der Spaltöffnungszellen sind resistenter.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.     

X-Ray Crystal Structure of a Highly Functionalized Thiophene as a New Backbone Amide Linker for Solid-phase Peptide Synthesis. Relationship between Crystal Structure and Reactivity

The development of new linkers (handles) for solid-phase synthesis provides new chemical opportunities for peptide synthesis. To understand the chemical properties of a recently developed backbone amide linker from a structural perspective, the crystal structure of S-((5-formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropionic acid (T-BAL2) was studied. Specifically, we wished to address whether this highly substituted thiophene retained planarity in the aromatic ring as well as between the aromatic ring and the aldehyde carbonyl. Furthermore, we sought an explanation for the relatively low reactivity in reductive aminations of the thienylaldehyde with amines in solution and on solid phase. Based on the crystal structure of T-BAL2, the thienyl-C (aldehyde) and C–O (aldehyde) bond lengths were applied as measures for the electron-deficiency (electrophilicity) of the aldehyde and compared to similar bond lengths found in previously reported formylated homo- and hetero-aromatic systems, which show significantly higher reactivity towards imine formation. The bond lengths found in the present structure are in accordance with normal C–C single bond and C–O double bond lengths. The high similarity in aldehyde bond lengths in the present system and in the reported systems indicates similar electron distribution in these systems. The lower reactivity of the present system may therefore not be attributed to electronic factors.     

Habitat selection by large mammals in a southern Brazilian Atlantic Forest

Habitat selection, which is mainly a consequence of competition and predation, allows species to coexist. The present study was conducted in two reserves in an Atlantic Forest area in Santa Catarina State, southern Brazil, and provided information on several large mammal species through photographic records. Records were related to certain environmental parameters, such as width of passages (trails and roads), vegetation density and proximity to water, in order to assess the relationship between each mammal species and its microhabitat. Thirty-two camera trap stations were placed during 17 months for 150.8 (±62.2) days on average. Terrestrial mammals tended to use different habitats: Puma concolor used mainly dirt roads and open areas; Leopardus pardalis, Cerdocyon thous and Nasua nasua used more large trails and intermediate-forested sites; and Cuniculus paca, Dasypus novemcinctus, Leopardus tigrinus, Eira barbara and Leopardus wiedii were recorded more often on narrow trails and in densely forested sites. Some of these forest species, such as D. novemcinctus, C. paca and L. pardalis, also showed relationships with watercourses. Information on habitat selection allows more effort to be addressed to the habitat associated with focal species, and indicates the significance of environmental heterogeneity, which makes it possible for species to coexist.     

Cytomegalovirus Infection in Inflammatory Bowel Disease Is Not Associated with Worsening of Intestinal Inflammatory Activity

Background

Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus.

Aim

Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations.

Methods

Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient.

Results

Among the 400 eligible patients, 249 had Crohn''s disease, and 151 had ulcerative colitis. In the group of Crohn''s disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine)

Conclusion

The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel disease activity.     

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage–host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.     

Frontal Lobe Hemodynamic Responses to Painful Stimulation: A Potential Brain Marker of Nociception

The purpose of this study was to use functional near-infrared spectroscopy (fNIRS) to examine patterns of both activation and deactivation that occur in the frontal lobe in response to noxious stimuli. The frontal lobe was selected because it has been shown to be activated by noxious stimuli in functional magnetic resonance imaging studies. The brain region is located behind the forehead which is devoid of hair, providing a relative ease of placement for fNIRS probes on this area of the head. Based on functional magnetic resonance imaging studies showing blood-oxygenation-level dependent changes in the frontal lobes, we evaluated functional near-infrared spectroscopy measures in response to two levels of electrical pain in awake, healthy human subjects (n = 10; male = 10). Each subject underwent two recording sessions separated by a 30-minute resting period. Data collected from 7 subjects were analyzed, containing a total of 38/36 low/high intensity pain stimuli for the first recording session and 27/31 pain stimuli for the second session. Our results show that there is a robust and significant deactivation in sections of the frontal cortices. Further development and definition of the specificity and sensitivity of the approach may provide an objective measure of nociceptive activity in the brain that can be easily applied in the surgical setting.     

Phthalates Are Metabolised by Primary Thyroid Cell Cultures but Have Limited Influence on Selected Thyroid Cell Functions In Vitro

Phthalates are plasticisers added to a wide variety of products, resulting in measurable exposure of humans. They are suspected to disrupt the thyroid axis as epidemiological studies suggest an influence on the peripheral thyroid hormone concentration. The mechanism is still unknown as only few in vitro studies within this area exist. The aim of the present study was to investigate the influence of three phthalate diesters (di-ethyl phthalate, di-n-butyl phthalate (DnBP), di-(2-ethylhexyl) phthalate (DEHP)) and two monoesters (mono-n-butyl phthalate and mono-(2-ethylhexyl) phthalate (MEHP)) on the differentiated function of primary human thyroid cell cultures. Also, the kinetics of phthalate metabolism were investigated. DEHP and its monoester, MEHP, both had an inhibitory influence on 3''-5''-cyclic adenosine monophosphate secretion from the cells, and MEHP also on thyroglobulin (Tg) secretion from the cells. Results of the lactate dehydrogenase-measurements indicated that the MEHP-mediated influence was caused by cell death. No influence on gene expression of thyroid specific genes (Tg, thyroid peroxidase, sodium iodine symporter and thyroid stimulating hormone receptor) by any of the investigated diesters could be demonstrated. All phthalate diesters were metabolised to the respective monoester, however with a fall in efficiency for high concentrations of the larger diesters DnBP and DEHP. In conclusion, human thyroid cells were able to metabolise phthalates but this phthalate-exposure did not appear to substantially influence selected functions of these cells.