Microbial Dissemination Research Articles

Microbial laden mobile phones from international conference attendees pose potential risks to public health and biosecurity

IntroductionMobile phones, contaminated with pathogenic microorganisms, have the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to international conference attendees may serve as a model for global microbial dissemination, posing potential risks to public health and biosecurity. AimThis study aimed to profile the microbes present on mobile phones belonging to delegates attending an international scientific conference through use of metagenomic shotgun DNA sequencing. MethodsTwenty mobile phones, representing ten different geographical zones from around the world, were swabbed and pooled together into ten geographical-specific samples for high definition next-generation DNA sequencing. WONCA council members were invited to participate and provided verbal consent. Following DNA extraction, next generation sequencing, to a depth of approximately 10Gbp per sample, was undertaken on a v1.5 Illumina NovaSeq6000 system. Bioinformatic analysis was performed via the CosmosID platform. ResultsA total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of 882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human pathogens, including ESKAPE and HACEK bacteria were found on mobile phones. Discussion/conclusionMobile phones of international attendees are contaminated with many & varied microorganisms. Further research is required to characterize the risks these devices pose for biosecurity and public health. Development of new policies which appropriately address and prevent such risks maybe warranted.

Interactions between prokaryotic polysaccharides and colloidal magnetic nanoparticles for bacteria removal: A strategy for circumventing antibiotic resistance

Highly stable, colloidal iron oxide nanoparticles with an oxyhydroxide-like surface were used as bacteria-capturing nano-baits. Peptidoglycan isolated from Listeria spp was used as bacteria polysaccharide model, and the nanoparticle binding was characterized showing a Langmuir isotherm constant, KL, equal to 50 ± 3 mL mg−1. The chemical affinity was further supported by dynamic light scattering, transmission electron microscopy, and infrared and UV–Vis data, pointing at the occurrence of extended, coordinative multiple point bindings. The interaction with Gram (+) (Listeria spp) and Gram (−) (Aeromonas veronii) bacteria was shown to be effective and devoid of any toxic effect. Moreover, a real sample, containing a population of several oligotrophic bacteria strains, was incubated with 1 g L−1 of nanoparticle suspension, in the absence of agitation, showing a 100 % capture efficiency, according to plate count. A nanoparticle regeneration method was developed, despite the known irreversibility of such bacterial-nanosurface binding, restoring the bacteria capture capability. This nanomaterial represents a competitive option to eliminate microbiological contamination in water as an alternative strategy to antibiotics, aimed at reducing microbial resistance dissemination. Finally, beyond their excellent features in terms of colloidal stability, binding performances, and biocompatibility this nanoparticle synthesis is cost effective, scalable, and environmentally sustainable.

In Vivo and in Vitro activity of colistin-conjugated bimetallic silver-copper oxide nanoparticles against Pandrug-resistant Pseudomonas aeruginosa

BackgroundAddressing microbial resistance urgently calls for alternative treatment options. This study investigates the impact of a bimetallic formulation containing colistin, silver, and copper oxide on a pandrug-resistant, highly virulent Pseudomonas aeruginosa (P. aeruginosa) isolate from a cancer patient at the National Cancer Institute, Cairo University, Egypt.MethodsSilver nanoparticles (Ag NPs), copper oxide nanoparticles (CuO NPs), and bimetallic silver-copper oxide nanoparticles (Ag-CuO NPs) were synthesized using gamma rays, combined with colistin (Col), and characterized by various analytical methods. The antimicrobial activity of Col-Ag NPs, Col-CuO NPs, and bimetallic Col-Ag-CuO NPs against P. aeruginosa was evaluated using the agar well diffusion method, and their minimum inhibitory concentration (MIC) was determined using broth microdilution. Virulence factors such as pyocyanin production, swarming motility, and biofilm formation were assessed before and after treatment with bimetallic Col-Ag-CuO NPs. The in vivo efficacy was evaluated using the Galleria mellonella model, and antibacterial mechanism were examined through membrane leakage assay.ResultsThe optimal synthesis of Ag NPs occurred at a gamma ray dose of 15.0 kGy, with the highest optical density (OD) of 2.4 at 375 nm. Similarly, CuO NPs had an optimal dose of 15.0 kGy, with an OD of 1.5 at 330 nm. Bimetallic Ag-CuO NPs were most potent at 15.0 kGy, yielding an OD of 1.9 at 425 nm. The MIC of colistin was significantly reduced when combined with nanoparticles: 8 µg/mL for colistin alone, 0.046 µg/mL for Col-Ag NPs, and 0.0117 µg/mL for Col-Ag-CuO NPs. Bimetallic Col-Ag-CuO NPs reduced the MIC four-fold compared to Col-Ag NPs. Increasing the sub-inhibitory concentration of bimetallic nanoparticles from 0.29 × 10-2 to 0.58 × 10-2 µg/mL reduced P. aeruginosa swarming by 32–64% and twitching motility by 34–97%. At these concentrations, pyocyanin production decreased by 39–58%, and biofilm formation was inhibited by 33–48%. The nanoparticles were non-toxic to Galleria mellonella, showing 100% survival by day 3, similar to the saline-treated group.ConclusionsThe synthesis of bimetallic Ag-CuO NPs conjugated with colistin presents a promising alternative treatment for combating the challenging P. aeruginosa pathogen in hospital settings. Further research is needed to explore and elucidate the mechanisms underlying the inhibitory effects of colistin-bimetallic Ag-CuO NPs on microbial persistence and dissemination.

Anthropogenic pollution may enhance natural transformation in water, favouring the spread of antibiotic resistance genes

Aquatic ecosystems are crucial in the antimicrobial resistance cycle. While intracellular DNA has been extensively studied to understand human activity's impact on antimicrobial resistance gene (ARG) dissemination, extracellular DNA is frequently overlooked. This study examines the effect of anthropogenic water pollution on microbial community diversity, the resistome, and ARG dissemination. We analyzed intracellular and extracellular DNA from wastewater treatment plant effluents and lake surface water by shotgun sequencing. We also conducted experiments to evaluate anthropogenic pollution's effect on transforming extracellular DNA (using Gfp-plasmids carrying ARGs) within a natural microbial community. Chemical analysis showed treated wastewater had higher anthropogenic pollution-related parameters than lake water. The richness of microbial community, antimicrobial resistome, and high-risk ARGs was greater in treated wastewaters than in lake waters both for intracellular and extracellular DNA. Except for the high-risk ARGs, richness was significantly higher in intracellular than in extracellular DNA. Several ARGs were associated with mobile genetic elements and located on plasmids. Furthermore, Gfp-plasmid transformation within a natural microbial community was enhanced by anthropogenic pollution levels. Our findings underscore anthropogenic pollution's pivotal role in shaping microbial communities and their antimicrobial resistome. Additionally, it may facilitate ARG dissemination through extracellular DNA plasmid uptake.

Heuristic approaches to risk assessment and prognosis of sepsis in dogs

This study examines approaches to assessing the risk of death and predicting the development of sepsis in dogs. Sepsis remains one of the key clinical problems in medicine and veterinary medicine, due to its widespread prevalence throughout the world, leading to high mortality. Sepsis is based on the reaction of systemic inflammation of the body, which is realized in the presence of signs of SIRS, bacteremia and/or an infectious focus of various nature in combination with acute signs of organ dysfunction and/or evidence of microbial dissemination. Since sepsis develops only progressively, it is impossible for the animal to recover independently without providing it with timely and appropriate medical care. Today, one of the key issues remains improving approaches to assessing the risk of death and predicting the development of sepsis in dogs. An attempt to combine the pathogenicity factors of an infectious agent and the failure of the host’s defense mechanisms is reflected in the concepts of PIRO, SAPS and MODS, which evaluate the links in the etiopathogenesis of sepsis. Thus, heuristic approaches will allow us to come closer to the development of pathogenetically based methods for assessing the risk of death of animals and predicting the development of sepsis in dogs. These approaches are based on criteria for assessing PIRO indicators: predisposition (P), including breed, sex, age of the animal; infection (I) associated with the type of pathogen detected; systemic response of the body to infection R, characterized by the main clinical indicators - body temperature, heart rate, respiratory rate and the number of leukocytes in the blood; organ dysfunction (O) identified in one or more organs of the animal. An important role in the objective prognosis is given to the evaluation criteria on the SAPS scale, taking into account changes in indicators towards hyperinflammation (SIRS) or immunosuppression (MODS), which determine the stage of development of sepsis and the likelihood of the risk of death of the sick animal. On average, the predictive accuracy of quantifying disease severity systems is 70–85%. An algorithm for translating clinical and laboratory data into a SAPS scale of indicators (scores) that determines the severity of the disease in leptospirosis in dogs is proposed. It has been shown that on the SAPS scale for canine leptospirosis, a score of 29

Survival of Salmonella Typhimurium in the hemolymph of the German cockroach vector is limited by both humoral immune factors and hemocytes but not by trehalose metabolism.

The German cockroach (Blattella germanica) has been linked to transmission of Salmonella enterica serovar Typhimurium (S. Typhimurium), but infection dynamics within this vector are poorly characterized. Our recent work has focused on S. Typhimurium infection in the cockroach gut. However, microbial dissemination to the hemolymph is an essential aspect of many vector-borne pathogen transmission cycles and could potentially contribute to S. Typhimurium colonization of cockroaches. Therefore, the goal of this study was to examine the ability of S. Typhimurium to disseminate, survive, and proliferate in the hemolymph of cockroaches after oral infection. We detected only low numbers of bacteria in the hemolymph of a minority of insects (~26%) after oral infection. Further, S. Typhimurium was unable to survive overnight in cell-free hemolymph. Several hypotheses to explain the inability of S. Typhimurium to colonize hemolymph were tested. First, we investigated the ability of S. Typhimurium to metabolize trehalose, the primary sugar in hemolymph. S. Typhimurium grew efficiently in vitro using trehalose as a sole carbon source and mutant strains lacking trehalose metabolism genes exhibited no growth deficiencies in media mimicking the composition of hemolymph, suggesting that trehalose metabolism ability is not a factor involved in restricting survival in hemolymph. On the other hand, heat-inactivated cell-free hemolymph was permissive of S. Typhimurium growth, demonstrating that survival in hemolymph is limited specifically by heat-labile humoral factors. The involvement of cellular immune responses was also investigated and cockroach hemocytes in culture were observed to internalize S. Typhimurium within 1 h of exposure. Most hemocytes harbored few to no bacteria after 24 h, indicating that hemocyte responses are additionally involved in clearing infection from the hemolymph. However, dense intracellular clusters of S. Typhimurium were observed sporadically, suggesting a small subset of hemocytes may serve as reservoirs for bacterial replication. Together, our results reveal that a minute proportion of ingested S. Typhimurium is able to escape the cockroach gut and enter the hemolymph, but this systemic population is limited by both humoral effectors and hemocytes. Thus, we conclude that invasion of the hemolymph appears minimally important for colonization of the cockroach vector and that colonization of the gut is the main driver of vector-borne transmission. Our insight into the antimicrobial mechanisms of cockroach hemolymph also highlights the strong ability of these prevalent pests/vectors to cope with frequent infectious challenges in septic habitats.

Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses.

Neutrophils display an array of biological functions including the formation of neutrophil extracellular traps (NETs), web-like structures specialized in trapping, neutralizing, killing and preventing microbial dissemination within the host. However, NETs contribute to a number of inflammatory pathologies, including severe equine asthma. Tamoxifen (TX) is a selective estrogen receptor modulator which belongs to the triphenylethyllenes group of molecules, and which is used as a treatment in all stages of estrogen-positive human breast cancer. Our previous results suggest that tamoxifen can modulate neutrophil functionality and promote resolution of inflammation; this would partly explain the clinical beneficial effect of this drug in horses with airway inflammation. Enhanced NETs production has been reported with tamoxifen use in humans, but minimal data exists regarding the drug's effect on NETs in horses. The aim of this study is to assess the in vitro effect of TX on NETs formation from peripheral blood of healthy horses. Five clinically healthy mixed-breed adult horses were enrolled in the study. For this, cellular free DNA quantification, immunofluorescence for the visualization of NETs, assessment of different types of NETs, and detection of mitochondrial superoxide. TX induced NETs formation at a concentration of 10 uM. Our results show that only two types of NETs were induced by TX: 95% spread NETs (sprNETs) and 5% aggregated NETs (aggNETs). Furthermore, induction of these NETs could be influenced by mitochondrial ROS. Future research should involve an In vivo study of horses with severe asthma and TX treatment, to evaluate BALF neutrophil NET formation. In conclusion, this in vitro study suggests that the resolution of inflammation by TX in horses with airway inflammation is due to inhibition of other neutrophilic functions but not to NET formation.

Emerging role of neutrophil extracellular traps in the complications of diabetes mellitus.

Immune dysfunction is widely regarded as one of the central tenants underpinning the pathophysiology of diabetes mellitus (DM) and its complications. When discussing immunity, the role of neutrophils must be accounted for: neutrophils are the most abundant of the circulating immune cells and are the first to be recruited to sites of inflammation, where they contribute to host defense via phagocytosis, degranulation, and extrusion of neutrophil extracellular traps (NETs). NETs are composed of DNA associated with nuclear and cytosolic neutrophil proteins. Although originally reported as an antimicrobial strategy to prevent microbial dissemination, a growing body of evidence has implicated NETs in the pathophysiology of various autoimmune and metabolic disorders. In these disorders, NETs propagate a pathologic inflammatory response with consequent tissue injury and thrombosis. Many diabetic complications—such as stroke, retinopathy, impaired wound healing, and coronary artery disease—involve these mechanisms. Therefore, in this review, we discuss laboratory and clinical data informing our understanding of the role of NETs in the development of these complications. NET markers, including myeloperoxidase, citrullinated histone H3, neutrophil elastase, and cell-free double-stranded DNA, can easily be measured in serum or be detected via immunohistochemical/immunocytochemical staining of tissue specimens. Therefore, NET constituents potentially constitute reliable biomarkers for use in the management of diabetic patients. However, no NET-targeting drug is currently approved for the treatment of diabetic complications; a candidate drug will require the outcomes of well-designed, robust clinical trials assessing whether NET inhibition can benefit patients in terms of morbidity, quality of life, health expenditures, and mortality. Therefore, much work remains to be done in translating these encouraging pieces of data into clinical trials for NET-targeting medications to be used in the clinic.

Relationship between maternal and/or newborn cholesterol levels and neonatal septicemia: protocol for a Ugandan cohort of mother-newborn pairs

BackgroundMany aspects of microbial dissemination appear to vary with host cholesterol levels. Since neonatal septicemia remains a leading cause of newborn admissions and mortality in resource-limited settings, the contribution of abnormal cholesterol levels in maternal and/or newborn blood to the risk of neonatal septicemia and outcome requires elucidation. We aim to determine a relationship between maternal serum and neonatal cord blood cholesterol levels and neonatal septicemia.MethodsThis will be a mother-newborn pair cohort study. Approximately 353 pregnant women who are eligible and consent to participate in the study will have blood drawn for a lipid profile. Upon delivery, we will analyse the cord blood cholesterol of their newborns and follow them for 28 days to determine whether the infants develop clinical signs and symptoms suggestive of neonatal septicemia. Relative risk will be used to determine the association between cholesterol and newborn septicemia. Poisson regression will be used to estimate the relative risk (with 95% confidence intervals) of developing septicemia.DiscussionFindings from our study will contribute evidence to support the inclusion of lipid profile screening for pregnant women and newborns. Our study will determine whether newborns with abnormal cholesterol or those born to mothers with abnormal cholesterol will require rigorous follow-up in neonatal clinics.

Mobile phones are hazardous microbial platforms warranting robust public health and biosecurity protocols

Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones. These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour. Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination. To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff. Twenty-six mobile phones of health care staff were swabbed. DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling was performed. Survey questionnaires were handed to the staff to collect information on mobile phone usage and users’ behaviours. Each of the 26 mobile phones of this study was contaminated with microbes with the detection of antibiotic resistance and virulent factors. Taken together the sum of microbes and genes added together across all 26 mobile phones totalised 11,163 organisms (5714 bacteria, 675 fungi, 93 protists, 228 viruses, 4453 bacteriophages) and 2096 genes coding for antibiotic resistance and virulent factors. The survey of medical staff showed that 46% (12/26) of the participants used their mobile phones in the bathroom. Mobile phones are vectors of microbes and can contribute to microbial dissemination and nosocomial diseases worldwide. As fomites, mobile phones that are not decontaminated may pose serious risks for public health and biosecurity.

Abstract 3043: Exploring the microbial landscape of NSCLC through TRACERx

Abstract Bacterial and viral species present in the human lung are associated with COPD (chronic obstructive pulmonary disease) exacerbations, lung cancer initiation and response to immunotherapy by impacting immune cell recruitment. Recent studies have identified microbiomes present in normal and tumour low-biomass samples. These microbes, potentially present within cancer and immune cells, exist in niches specific to tumour types. These studies are often single-region and single-timepoint biopsy studies which promotes the question as to whether heterogeneity of the microbiome can exist within a tumour as well as progression of the microbiome from primary tumours to metastasis. Exploring microbial heterogeneity by leveraging the multi-region and longitudinal data from TRACERx, we aimed to identify microbes present clonally as well as studying enrichment of microbes in metastatic regions or associations with genotypes. We conducted 16S (V3V4) sequencing of over 600 TRACERx samples including primary, adjacent normal and metastatic tissues. Stringent decontamination was applied using computational methods as well as completed at sequencing centre and extraction level sites. The 16S data was aligned to the SILVA-138 reference genome using the QIIME2 pipeline. We also performed analysis on matched RNA-sequencing data through tools such as KRAKEN2 and SHOGUN. Diversity metrics across histological subtypes, COPD status or gender did not yield significant differences or show an enrichment of genus with patient characteristics. However, a trend of decreasing microbial diversity was associated with smoking which demonstrates that the effects of smoking can lead to dysbiosis of a healthy lung microbiome. Within primary tumours, increased tumour sampling improved the detection of microbial diversity and presence of microbes found at a lower abundance, revealing a heterogenous microbial landscape. Furthermore, matched primary and metastatic tumours are highly similar compared to tumours from unmatched patients (P = 0.017) This suggests that primary and metastatic tissues share a similar community within patients, however an enrichment of opportunistic microbes was found in metastatic tissue. Finally, we also measured the association of specific microbes with different driver genotypes and several significant associations were discovered such as NOTCH1+ LUSC (lung squamous cell carcinoma) patients were enriched for Firmicutes. Studying the microbial landscape of NSCLC through an evolutionary lens provides insight into microbial dissemination from primary tumours to metastases and co-occurrence with genotypes. The multi-region and longitudinal approach to studying the microbiome has exposed levels of microbial intratumor heterogeneity. Moreover, recognising that metastatic tumours have an overlapping microbiome to the primary site offers potential for durable therapeutic utility, via targeting clonally present species. Citation Format: Krupa Thakkar, Hongchang Fu, Selvaraju Veeriah, Kevin Litchfield, Charles Swanton, TRACERx Consortium. Exploring the microbial landscape of NSCLC through TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3043.

Bacteriophages vehiculate a high amount of antibiotic resistance determinants of bacterial origin in the Orne River ecosystem.

Aquatic environments are important dissemination routes of antibiotic resistance genes (ARGs) from and to pathogenic bacteria. Nevertheless, in these complex matrices, identifying and characterizing the driving microbial actors and ARG dissemination mechanisms they are involved in remain difficult. We here explored the distribution/compartmentalization of a panel of ARGs and mobile genetic elements (MGEs) in bacteria and bacteriophages collected in the water, suspended material and surface sediments from the Orne River ecosystem (France). By using a new bacteriophage DNA extraction method, we showed that, when packaging bacterial DNA, bacteriophages rather encapsidate both ARGs and MGEs than 16S rRNA genes, i.e. chromosomal fragments. We also show that the bacteria and bacteriophage capsid contents in ARGs/MGEs were similarly influenced by seasonality but that the distribution of ARGs/MGEs between the river physical compartments (water vs. suspended mater vs. sediment) is more impacted when these markers were carried by bacteria. These demonstrations will likely modify our understanding of the formation and fate of transducing viral particles in the environment. Consequently, they will also likely modify our estimations of the relative frequencies of the different horizontal gene transfer mechanisms in disseminating antibiotic resistance by reinforcing the roles played by environmental bacteriophages and transduction.

Metagenomic Sequencing and Reverse Transcriptase PCR Reveal That Mobile Phones and Environmental Surfaces Are Reservoirs of Multidrug-Resistant Superbugs and SARS-CoV-2.

BackgroundMobile phones of healthcare workers (HCWs) can act as fomites in the dissemination of microbes. This study was carried out to investigate microbial contamination of mobile phones of HCWs and environmental samples from the hospital unit using a combination of phenotypic and molecular methods.MethodsThis point prevalence survey was carried out at the Emergency unit of a tertiary care facility. The emergency unit has two zones, a general zone for non-COVID-19 patients and a dedicated COVID-19 zone for confirmed or suspected COVID-19 patients. Swabs were obtained from the mobile phones of HCWs in both zones for bacterial culture and shotgun metagenomic analysis. Metagenomic sequencing of pooled environmental swabs was conducted. RT-PCR for SARS-CoV-2 detection was carried out.ResultsBacteria contamination on culture was detected from 33 (94.2%) mobile phones with a preponderance of Staphylococcus epidermidis (n/N = 18/35), Staphylococcus hominis (n/N = 13/35), and Staphylococcus haemolyticus (n/N = 7/35). Two methicillin-sensitive and three methicillin-resistant Staphylococcus aureus, and one pan-drug-resistant carbapenemase producer Acinetobacter baumannii were detected. Shotgun metagenomic analysis showed high signature of Pseudomonas aeruginosa in mobile phone and environmental samples with preponderance of P. aeruginosa bacteriophages. Malassezia and Aspergillus spp. were the predominant fungi detected. Fourteen mobile phones and one environmental sample harbored protists. P. aeruginosa antimicrobial resistance genes mostly encoding for efflux pump systems were detected. The P. aeruginosa virulent factor genes detected were related to motility, adherence, aggregation, and biofilms. One mobile phone from the COVID-19 zone (n/N = 1/5; 20%) had positive SARS-CoV-2 detection while all other phone and environmental samples were negative.ConclusionThe findings demonstrate that mobile phones of HCWs are fomites for potentially pathogenic and highly drug-resistant microbes. The presence of these microbes on the mobile phones and hospital environmental surfaces is a concern as it poses a risk of pathogen transfer to patients and dissemination into the community.

Neutrophil extracellular traps in chronic lung disease: implications for pathogenesis and therapy.

Neutrophilic inflammation has a key role in the pathophysiology of multiple chronic lung diseases. The formation of neutrophil extracellular traps (NETs) has emerged as a key mechanism of disease in neutrophilic lung diseases including asthma, COPD, cystic fibrosis and, most recently, bronchiectasis. NETs are large, web-like structures composed of DNA and anti-microbial proteins that are able to bind pathogens, prevent microbial dissemination and degrade bacterial virulence factors. The release of excess concentrations of proteases, antimicrobial proteins, DNA and histones, however, also leads to tissue damage, impaired mucociliary clearance, impaired bacterial killing and increased inflammation. A number of studies have linked airway NET formation with greater disease severity, increased exacerbations and overall worse disease outcomes across the spectrum of airway diseases. Treating neutrophilic inflammation has been challenging in chronic lung disease because of the delicate balance between reducing inflammation and increasing the risk of infections through immunosuppression. Novel approaches to suppressing NET formation or the associated inflammation are in development and represent an important therapeutic target. This review will discuss the relationship between NETs and the pathophysiology of cystic fibrosis, asthma, COPD and bronchiectasis, and explore the current and future development of NET-targeting therapies.

INTERLEUKIN-17 RECEPTOR SIGNALING TO PANETH CELLS HELPS MAINTAIN INTESTINAL INTEGRITY AFTER GAMMA RADIATION INDUCED INJURY

Abstract BACKGROUND IL-17A/F signaling through the IL-17RA/RC receptor complex plays a vital role in immunity and inflammation. The expression of IL-17RA/RC by diverse intestinal epithelial cell types including Paneth cells and the observation that therapies neutralizing IL-17A or IL-17RA in Crohn’s disease (CD) patients induce adverse events suggest essential homeostatic functions of this cytokine. Paneth cells and their antimicrobial products (a-defensins, lysozyme etc.) play a critical role in mediating small intestinal host defense under homeostatic conditions as well as after injury or infection, and their dysregulation constitutes a pathogenic factor for CD. The specific role of intestinal IL-17A signaling in Paneth cells to regulate host defense at steady state or after intestinal inflammation remains poorly understood. Thus, we hypothesize that intestinal IL-17A signaling, specifically in Paneth cells, plays an important role in regulating microbiota colonization as well as maintaining intestinal integrity after gamma radiation induced injury. METHODS To examine the importance of IL-17RA signaling to Paneth cells, we utilized total, entire gut epithelium (Il17rafl/fl;villin-cre) and Paneth cell-specific IL-17RA knockout mice (Il17rafl/fl;Defa6-cre+). We subjected these mice with gamma irradiation (1200 cGy) to study intestinal injury and regenerative responses. Mice were sacrificed 3/5 days post irradiation. Terminal ilium was collected from naïve or irradiated mice for immunofluorescence imaging, RT-PCR, RNA sequencing, and enteroid cultures. RESULTS Our preliminary data from irradiated Il17ra-/- and Il17rafl/fl;villin-cre mice suggest that IL-17A signaling is important in maintaining intestinal barrier integrity. Specifically, these mice revealed increased microbial dissemination to liver and spleen compared to their respective control mice. Reduced expression of Lyz1 3 days post radiation in the terminal ileum of Il17ra-/- mice suggest impaired Paneth cell function. Cessation of Paneth cell specific IL-17RA signaling, also resulted in systemic bacterial dissemination 5 days post radiation. We also observed increased level of fecal Lcn2 in irradiated Il17rafl/fl;Defa6-cre+ mice. To understand Paneth cell-specific IL-17RA functions, we performed 16S microbial and RNA sequencing from ileum luminal contents and terminal ilea of naïve Il17rafl/fl;Defa6-cre+/- mice, respectively. While microbial 16s sequencing revealed no change to the overall microbial community, specific changes were observed at a select family level. Specifically, the level of Streptococcaceae was increased. RNA sequencing data showed no difference in Paneth cell antimicrobial peptides but did show reduced Caspase 4 expression in Il17rafl/fl;Defa6-cre+ mice. Collectively, our data revealed an important role of IL-17RA signaling in Paneth cell function at steady state and after injury.

Postnatal Sepsis and Bronchopulmonary Dysplasia in Premature Infants: Mechanistic Insights into "New BPD".

Bronchopulmonary dysplasia (BPD) is a debilitating disease in premature infants resulting from lung injury that disrupts alveolar and pulmonary vascular development. Despite the use of lung-protective ventilation and targeted oxygen therapy, BPD rates have not significantly changed over the last decade. Recent evidence suggests that sepsis and conditions initiating the systemic inflammatory response syndrome in preterm infants are key risk factors for BPD. However, the mechanisms by which sepsis-associated systemic inflammation and microbial dissemination program aberrant lung development are not fully understood. Progress has been made within the last 5 years with the inception of animal models allowing mechanistic investigations into neonatal acute lung injury and alveolar remodeling attributable to endotoxemia and necrotizing enterocolitis. These recent studies begin to unravel the pathophysiology of early endothelial immune activation via pattern recognition receptors such as Toll-like receptor 4 and disruption of critical lung developmental processes such as angiogenesis, extracellular matrix deposition, and ultimately alveologenesis. Here we review scientific evidence from preclinical models of neonatal sepsis-induced lung injury to new data emerging from clinical literature.

A Fragile Balance: Does Neutrophil Extracellular Trap Formation Drive Pulmonary Disease Progression?

Neutrophils act as the first line of defense during infection and inflammation. Once activated, they are able to fulfil numerous tasks to fight inflammatory insults while keeping a balanced immune response. Besides well-known functions, such as phagocytosis and degranulation, neutrophils are also able to release “neutrophil extracellular traps” (NETs). In response to most stimuli, the neutrophils release decondensed chromatin in a NADPH oxidase-dependent manner decorated with histones and granule proteins, such as neutrophil elastase, myeloperoxidase, and cathelicidins. Although primarily supposed to prevent microbial dissemination and fight infections, there is increasing evidence that an overwhelming NET response correlates with poor outcome in many diseases. Lung-related diseases especially, such as bacterial pneumonia, cystic fibrosis, chronic obstructive pulmonary disease, aspergillosis, influenza, and COVID-19, are often affected by massive NET formation. Highly vascularized areas as in the lung are susceptible to immunothrombotic events promoted by chromatin fibers. Keeping this fragile equilibrium seems to be the key for an appropriate immune response. Therapies targeting dysregulated NET formation might positively influence many disease progressions. This review highlights recent findings on the pathophysiological influence of NET formation in different bacterial, viral, and non-infectious lung diseases and summarizes medical treatment strategies.

Experimental and modelling evidence of splash effects on manure borne Escherichia coli washoff.

In tropical montane South-East Asia, recent changes in land use have induced increased runoff, soil erosion and in-stream suspended sediment loads. Land use change is also contributing to increased microbial pathogen dissemination and contamination of stream waters. Escherichia coli (E. coli) is frequently used as an indicator of faecal contamination. Field rain simulations were conducted to examine how E. coli is exported from the surface of upland, agricultural soils during runoff events. The objectives were to characterize the loss dynamics of this indicator from agricultural soils contaminated with livestock waste, and to identify the effect of splash on washoff. Experiments were performed on nine 1 m2 plots, amended or not with pig or poultry manure. Each plot was divided into two 0.5 m2 sub-plots. One of the two sub-plots was protected with a mosquito net for limiting the raindrop impact effects. Runoff, soil detachment by raindrop impact and its entrainment by runoff, and E. coli loads and discharge were measured for each sub-plot. The results show that raindrop impact strongly enhances runoff generation, soil detachment and entrainment and E. coli export. When the impact of raindrops was reduced with a mosquito net, total runoff was reduced by more than 50%, soil erosion was on average reduced by 90% and E. coli export from the amended soil surface was on average 3 to 8 times lower. A coupled physics-based approach was performed using the Cast3M platform for modelling the time evolutions of runoff, solid particles detachment and transfer and bacteria transport that were measured for one of the nine plots. After estimation of the saturated hydraulic conductivity, soil erodibility and attachment rate of bacteria, model outputs were consistent with measured runoff coefficients, suspended sediment and E. coli loads. This work therefore underlines the need to maintain adequate vegetation at the soil surface to avoid the erosion and export of soil borne potential pathogens towards downstream aquatic systems.

ИЗМЕНЕНИЕ ФИЗИКО-ХИМИЧЕСКИХ СВОЙСТВ ЖЕЛЧИ В ПРОЦЕССЕ ВОЗНИКНОВЕНИЯ И МАНИФЕСТАЦИИ ХОЛАНГИТА. ОСТРЫЙ ХОЛАНГИТ: ВОЗМОЖНЫЕ МЕХАНИЗМЫ ЗАПУСКА

Objective. To determine the possible triggering mechanism for the development and manifestation of acute suppurative cholangitis based on the study of the rheological, physical and chemical properties of bile. Methods. The bile of choledoch was studied inpatients (n=41) with calculous obstruction of the biliary tract, 25 of them had clinical picture of cholangitis. For comparison, 32 patients with asymptomatic choledocholithiasis were examined. Bile sampling was performed in each patient twice: the first bile intake was obtained during duodenoscopy and cannulation of the major papilla of the duodenum and endoscopic papillosphincterotomy and the second portion - during a follow-up examination three days after the endoscopic papillotomy. The biliary pH, viscosity, the amount of solids in it, the concentration of primary bile acids, microbial contamination and the intraductal pressure were studied. Results. For the first time, it has been hypothesized that a reduced concentration of bile acids in the bile causes a subsequent chain of triggering and manifestation of acute cholangitis. In the first bile intake the concentration of bile acids is three fold decreased in patients with acute cholangitis in comparison with the patients of control group, and in the second case it reliable increased but was lower than in the control group. It was a small amount of bile acids caused sedimentation of a colloidal bile solution, increased its viscosity and amount of dry sediment, caused precipitation and sludge. This chain process caused the instantaneous inclusion of concretion in sludge and acute obstruction of the choledoch. As a result of bacterial contamination, the microbial dissemination of bile increases by 10<sup>3</sup>-10<sup>4</sup> times, the choledoch becomes like an abscess. Endoscopic papillosphincterotomy breaks this chain and surgical management is the definitive mode of treatment for decompression and biliary track sanitation. Conclusion. A decrease of bile acids in the content of the bile causes a chain process of sedimentation, the formation of sludge and a complete block of the choledoch, which is the primary trigger for cholangitis. What this paper adds Scientific and theoretical principles which in their essencedetermine the possible mechanism of the development of cholangitis due to choledocholithiasis have been developed. The physicochemical properties of bile play a decisive role in the triggering mechanism of cholangitis. Reduction of the synthesis of bile acids in the liver or their resorption in the intestine starts an avalanche-like process of sedimentation in the micellar solution of bile in the choledoch, sludge with the stones appears, the pH decreases, intraductal pressure rises, as a result of bacterial infection microbial contamination the microbal dissemination increases by 10<sup>3</sup>-10<sup>4</sup> fold.

In Vitro Identification and Isolation of Human Neutrophil Extracellular Traps.

Neutrophils release web like-structures known as neutrophil extracellular traps (NETs) that ensnare and kill microorganisms. These networks are constituted of a DNA scaffold with associated antimicrobial proteins, which are released to the extracellular space as an effective mechanism to fight against invading microorganisms. In parallel with this beneficial role to avoid microbial dissemination and wall off infections, accumulating evidence supports that under certain circumstances, NETs can exert deleterious effects in inflammatory, autoimmune, and thrombotic pathologies. Research on NET properties and their role in pathophysiological processes is a rapidly evolving and expanding field. Here, we describe a combination of methods to achieve a successful in vitro NET visualization, semiquantification, and isolation.