Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, ...thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype-genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
As a significant cause of global morbidity and mortality, Clostridioides difficile infections (CDIs) are listed by the Centres for Disease Control and prevention as one of the top 5 urgent threats in ...the USA. CDI occurs from gut microbiome dysbiosis, typically through antibiotic-mediated disruption; however, antibiotics are the treatment of choice, which can result in recurrent infections. Here, we highlight new treatments available and provide a perspective on different classes of future treatments.
Due to the reduced risk of disease recurrence, the microbiome-sparing antibiotic Fidaxomicin has been recommended as the first-line treatment for C. difficile infection. Based on the success of faecal microbiota transplantations (FMT) in treating CDI recurrence, defined microbiome biotherapeutics offer a safer and more tightly controlled alterative as an adjunct to antibiotic therapy. Given the association between antibiotic-mediated dysbiosis of the intestinal microbiota and the recurrence of CDI, future prospective therapies aim to reduce the dependence on antibiotics for the treatment of CDI.
With current first-in-line antibiotic therapy options associated with high levels of recurrent CDI, the availability of new generation targeted therapeutics can really impact treatment success. There are still unknowns about the long-term implications of these new CDI therapeutics, but efforts to expand the CDI treatment toolbox can offer multiple solutions for clinicians to treat this multifaceted infectious disease to reduce patient suffering.
LOV-based reporters for fluorescence imaging Buckley, Anthony M; Petersen, Jan; Roe, Andrew J ...
Current opinion in chemical biology,
August 2015, 2015-Aug, 2015-08-00, 20150801, Letnik:
27
Journal Article
Recenzirano
Odprti dostop
•Flavin-binding LOV domains have been engineered as green fluorescent reporters.•Several LOV-based FPs are available with different photophysical properties.•LOV-based FPs can circumvent limitations ...commonly associated with GFP-related FPs.•LOV fluorescence has been exploited for various imaging and biosensor applications.•Their properties also offer new possibilities in cancer therapy and optogenetics.
Chromophore-binding domains from plant and bacterial photoreceptor proteins have recently gathered increasing attention as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (light, oxygen, or voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size, pH and thermal stability, utility under anaerobic conditions and their ability to generate reactive oxygen species. This review focuses on the potential applications of this emerging class of fluorescent reporters, discusses the advantages and limitations of LOV-based FPs, whilst offering insights regarding the further development of this technology for bioimaging and photodynamic therapy.
Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid ...evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers ...target specificity and dose amplification in situ, but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis of C. difficile in vitro and prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reduced C. difficile colonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI.
Can SARS-CoV-2 be transmitted via faeces? Moura, Ines B; Buckley, Anthony M; Wilcox, Mark H
Current opinion in gastroenterology,
01/2022, Letnik:
38, Številka:
1
Journal Article
Recenzirano
Odprti dostop
COVID-19 patients can present gastrointestinal symptoms, being diarrhoea one of the most frequent, suggesting intestinal health can be impacted by COVID-19. Here, we will discuss whether there is a ...correlation between the presence of SARS-CoV-2 RNA in faeces and diarrhoea, the relevance of gastrointestinal symptoms in disease diagnosis and transmission, and how COVID-19 can impact the gut microbial balance.
SARS-CoV-2 RNA has been reported in faeces or rectal swabs of COVID-19 patients with and without diarrhoea, suggesting faecal shedding can occur independently of gastrointestinal symptoms. However, the presence of the virus in the intestine can persist beyond its presence in the respiratory tract, with some reports suggesting that SARS-CoV-2 in the faeces can be infectious.COVID-19 can impact the gut microbiota causing an enhancement of biosynthesis pathways that favour the expansion of bacterial pathogens in the inflamed gut, and causing a decline in commensals involved in the human immune response.
Gastrointestinal symptoms may be the first indication of COVID-19. SARS-CoV-2 in faeces can potentiate routes of disease transmission, particularly as the high viral loads reported in patients with severe illness suggest virus replication in the intestine may be possible.
There is a paradox when treating Clostridium difficile infection (CDI); treatment antibiotics reduce C. difficile colonization but cause further microbiota disruption and can lead to recurrent ...disease. The success of faecal microbiota transplants (FMT) in treating CDI has become a new research area in microbiome restorative therapies but are they a viable long-term treatment option?
C. difficile displays metabolic flexibility to use different nutritional sources during CDI. Using microbiome therapies for the efficient restoration of bile homeostasis and to reduce the bioavailability of preferential nutrients will target the germination ability of C. difficile spores and the growth rate of vegetative cells. Several biotechnology companies have developed microbiome therapeutics for treating CDI, which are undergoing clinical trials.
There is confidence in using restorative microbiome therapies for treating CDI after the demonstrated efficacy of FMT, where several biotechnology companies are aiming to supply what would be a 'first in class' treatment option. Efficient removal of C. difficile from the different intestinal biogeographies should be considered in future microbiome therapies. With the gut microbiota implicated in different diseases, more work is needed to assess the long-term consequences of microbiome therapies.
There is a medical need for antibacterial agents that do not damage the resident gut microbiota or promote the spread of antibiotic resistance. We recently described a prototypic precision ...bactericidal agent, Av-CD291.2, which selectively kills specific
strains and prevents them from colonizing mice. We have since selected two Av-CD291.2-resistant mutants that have a surface (S)-layer-null phenotype due to distinct point mutations in the
gene. Using newly identified bacteriophage receptor binding proteins for targeting, we constructed a panel of Avidocin-CDs that kills diverse
isolates in an S-layer sequence-dependent manner. In addition to bacteriophage receptor recognition, characterization of the mutants also uncovered important roles for S-layer protein A (SlpA) in sporulation, resistance to innate immunity effectors, and toxin production. Surprisingly, S-layer-null mutants were found to persist in the hamster gut despite a complete attenuation of virulence. These findings suggest antimicrobials targeting virulence factors dispensable for fitness in the host force pathogens to trade virulence for viability and would have clear clinical advantages should resistance emerge. Given their exquisite specificity for the pathogen, Avidocin-CDs have substantial therapeutic potential for the treatment and prevention of
infections.
Objective To evaluate the clinical safety of antenatal and postnatal N-acetylcysteine (NAC) as a neuroprotective agent in maternal chorioamnionitis in a randomized, controlled, double-blinded trial. ...Study design Twenty-two mothers >24 weeks gestation presenting within 4 hours of diagnosis of clinical chorioamnionitis were randomized with their 24 infants to NAC or saline treatment. Antenatal NAC (100 mg/kg/dose) or saline was given intravenously every 6 hours until delivery. Postnatally, NAC (12.5-25 mg/kg/dose, n = 12) or saline (n = 12) was given every 12 hours for 5 doses. Doppler studies of fetal umbilical and fetal and infant cerebral blood flow, cranial ultrasounds, echocardiograms, cerebral oxygenation, electroencephalograms, and serum cytokines were evaluated before and after treatment, and 12, 24, and 48 hours after birth. Magnetic resonance spectroscopy and diffusion imaging were performed at term age equivalent. Development was followed for cerebral palsy or autism to 4 years of age. Results Cardiovascular measures, cerebral blood flow velocity and vascular resistance, and cerebral oxygenation did not differ between treatment groups. Cerebrovascular coupling was disrupted in infants with chorioamnionitis treated with saline but preserved in infants treated with NAC, suggesting improved vascular regulation in the presence of neuroinflammation. Infants treated with NAC had higher serum anti-inflammatory interleukin-1 receptor antagonist and lower proinflammatory vascular endothelial growth factor over time vs controls. No adverse events related to NAC administration were noted. Conclusions In this cohort of newborns exposed to chorioamnionitis, antenatal and postnatal NAC was safe, preserved cerebrovascular regulation, and increased an anti-inflammatory neuroprotective protein. Trial registration ClinicalTrials.gov : NCT00724594.
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