Role of gut microbiota and bacterial translocation in acute intestinal injury and mortality in patients admitted in ICU for septic shock

IntroductionSepsis is a life-threatening organ dysfunction with high mortality rate. The gut origin hypothesis of multiple organ dysfunction syndrome relates to loss of gut barrier function and the ensuing bacterial translocation. The aim of this study was to describe the evolution of gut microbiota in a cohort of septic shock patients over seven days and the potential link between gut microbiota and bacterial translocation.MethodsSixty consecutive adult patients hospitalized for septic shock in intensive care units (ICU) were prospectively enrolled. Non-inclusion criteria included patients with recent or scheduled digestive surgery, having taken laxatives, pre- or probiotic in the previous seven days, a progressive digestive neoplasia, digestive lymphoma, chronic inflammatory bowel disease, moribund patient, and pregnant and lactating patients. The primary objective was to evaluate the evolution of bacterial diversity and richness of gut microbiota during seven days in septic shock. Epidemiological, clinical and biological data were gathered over seven days. Gut microbiota was analyzed through a metagenomic approach. 100 healthy controls were selected among healthy blood donors for reference basal 16S rDNA values.ResultsSignificantly lower bacterial diversity and richness was observed in gut microbiota of patients at Day 7 compared with Day 0 (p<0.01). SOFA score at Day 0, Acute Gastrointestinal Injury (AGI) local grade, septic shock origin and bacterial translocation had an impact on alpha diversity. A large increase in Enterococcus genus was observed at Day 7 with a decrease in Enterobacterales, Clostridiales, Bifidobacterium and other butyrate-producing bacteria.DiscussionThis study shows the importance of bacterial translocation during AGI in septic shock patients. This bacterial translocation decreases during hospitalization in ICUs in parallel to the decrease of microbiota diversity. This work highlights the role of gut microbiota and bacterial translocation during septic shock

The microbiome of diabetic foot ulcers and the role of biofilms

Diabetic Foot Ulcers are a common precursor to the development of infection and amputations. A breach in the protective skin barrier represents a portal of entry for invading microorganisms, where infective episodes frequently pursue. Three key areas that may augment clinical care are one. understanding what microorganisms are present in Diabetic Foot Ulcers, two. differentiating if microorganisms are planktonic microbial cells or slow growing microbial biofilms and three. treating Diabetic Foot Ulcers complicated by microorganisms with effective topical agents. As part of this thesis, 16S rDNA next generation sequencing was utilised to profile the microbiota of infected Diabetic Foot Ulcers (DFUs). Clinical / laboratory data and treatment outcomes were collected and correlated against microbiota data. Thirty-nine patients with infected DFUs were recruited over twelve-months. Shorter duration DFUs (less than six weeks) all had one dominant bacterial species (n= five of five, 100%, p <⋅001), S. aureus in three cases and S. agalactiae in two. Longer duration DFUs (≥six weeks) were diversely polymicrobial (p = .01) with an average of 63 (range 19-125) bacterial species. Severe Diabetic Foot Infections (DFIs) had complex microbiota’s and were distinctly dissimilar to less severe infections (p = .02), characterised by the presence of low frequency microorganisms. Our results confirm that short DFUs have a simpler microbiota’s consisting of pyogenic cocci but chronic DFUs have a highly polymicrobial microbiota. The duration of a DFU may be useful as a guide to directing antimicrobial therapy. Secondly, we utilised Scanning electron microscopy (SEM) and Fluorescent in situ Hybridisation (FISH) techniques to determine if DFUs were complicated by sessile, slow growing bacteria referred to as biofilms. 65 DFU specimens were obtained from subjects with infected chronic ulcers. Of the 65 DFU specimens evaluated by microscopy, all were characterized as containing biofilm (100%, p < .001). Molecular analyses of DFU specimens revealed diverse polymicrobial communities. No clinical visual cues were identified in aiding clinicians identify wound biofilm. Microscopy visualization when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and a paradigm shift of managing these complicated wounds needs to consider anti-biofilm strategies. Lastly, the effectiveness of various topical antimicrobials commonly used in woundcare were tested in two separate studies by employing in vitro models, ex vivo porcine skin explant models and in vivo human studies. In the first study, 17 participants with chronic non-healing DFUs due to suspected biofilm involvement were recruited to receive one-week application of Cadexomer Iodine ointment. Real-time qPCR was used to determine the microbial load with 11 participants exhibiting one-two Log10 reductions in microbial load after treatment, in comparison to six patients who experienced less than one log10 reduction (p =.04). Scanning electron microscopy (SEM) and/or fluorescent in situ hybridisation (FISH) confirmed the presence or absence of biofilm in all 17 participants. 16SrDNAnextgenerationsequencing provided useful insights that these wounds support complex polymicrobial communities and demonstrated that Cadexomer Iodine had a broad level of antimicrobial activity in reducing both facultative anaerobes such as Staphylococcus spp., Serratia spp., aerobes including Pseudomonas spp., and obligate anaerobes including Clostridiales family XI. In the second study, a range of topical antimicrobial wound solutions were tested under three different conditions; (in vitro) 4 % w/v melaleuca oil, polyhexamethylene biguanide, chlorhexidine, povidone iodine and hypochlorous acid were tested at short duration exposure times for 15-minutes against three-day mature biofilms of S. aureus and P. aeruginosa. (ex vivo) Hypochlorous acid was tested in a porcine skin explant model with twelve cycles of tenminute exposure, over 24 hours, against three-day mature P. aeruginosa biofilms. (in vivo) 4 % w/v Melaleuca Oil was applied for 15-minutes exposure, daily, for seven days, in ten patients with chronic non-healing Diabetic Foot Ulcers (DFUs) complicated by biofilm. In vitro assessment demonstrated variable efficacy in reducing biofilms ranging between 0.5 log10 reductions to full eradication. Repeated instillation of hypochlorous acid in a porcine model achieved less than one log10 reduction (0.77 log10, p < 0.1). Application of 4 % w/v melaleuca oil in vivo, resulted in no change to the total microbial load of DFUs complicated by biofilm (median log10 microbial load pre-treatment = 4.9 log10 versus 4.8 log10 (p = .43). In conclusion, to the best of our knowledge, the in vivo human studies testing the performances of topical antimicrobials represents the first in vivo evidence employing a range of molecular and microscopy techniques. These demonstrate the ability of Cadexomer Iodine (sustained release over 48-72 hours) to reduce the microbial load of chronic non-healing DFUs complicated by biofilm. In contrast, short durations of exposure to topical antimicrobial wound solutions commonly utilised by clinicians are ineffective against microbial biofilms, particularly when used in vivo

Propolis potentiates the effect of cranberry (Vaccinium macrocarpon) in reducing the motility and the biofilm formation of uropathogenic Escherichia coli.

One strategy to prevent urinary tract infections is the use of natural products such as cranberry (Vaccinium macrocarpon) and propolis. The objective of this study was to evaluate the impact of these products alone and combined on the motility and biofilm formation of a collection of representative uropathogenic Escherichia coli (UPEC). Motility was evaluated by the swarming and swimming capacity of the isolates in presence/absence of cranberry ± propolis. Early and late biofilm formation was observed with the Biofilm Ring test (BioFilm Control) and the crystal violet method. Cranberry alone was seen to have a variable effect on motility and biofilm formation unrelated to bacterial characteristics, but a reduced motility and biofilm formation was observed for all the isolates in the presence of cranberry + propolis. These results suggest that cranberry alone doesn't work on all the E. coli strains and propolis potentiates the effect of cranberry on UPEC, representing a new strategy to prevent recurrent urinary tract infections

Analysis of Microbial Communities: An Emerging Tool in Forensic Sciences

International audienceThe objective of forensic sciences is to find clues in a crime scene in order to reconstruct the scenario. Classical samples include DNA or fingerprints, but both have inherent limitations and can be uninformative. Another type of sample has emerged recently in the form of the microbiome. Supported by the Human Microbiome Project, the characteristics of the microbial communities provide real potential in forensics. They are highly specific and can be used to differentiate and classify the originating body site of a human biological trace. Skin microbiota is also highly specific and different between individuals, leading to its possibility as an identification tool. By extension, the possibilities of the microbial communities to be deposited on everyday objects has also been explored. Other uses include the determination of the post-mortem interval or the analysis of soil communities. One challenge is that the microbiome changes over time and can be influenced by many environmental and lifestyle factors. This review offers an overview of the main methods and applications to demonstrate the benefit of the microbiome to provide forensically relevant information

New Molecular Techniques to Study the Skin Microbiota of Diabetic Foot Ulcers

International audienceSignificance: Diabetic foot ulcers (DFU) are a major and growing public health problem. They pose difficulties in clinical practice in both diagnosis and management. Bacterial interactions on the skin surface are important in the pathophysiology of DFU and may contribute to a delay in healing. Fully identifying bacteria present in these wounds is difficult with traditional culture methods. New molecular tools, however, have greatly contributed to our understanding of the role of the cutaneous microbiota in DFU. Recent Advances: Molecular technologies revealed new information concerning how bacteria are organized in DFU. This has led to the concept of "functionally equivalent pathogroups," meaning that certain bacterial species which are usually nonpathogenic (or at least incapable of maintaining a chronic infection on their own) may coaggregate symbiotically in a pathogenic biofilm and act synergistically to cause a chronic infection. The distribution of pathogens in multispecies biofilms is nonrandom. The high bacterial diversity is probably related to the development of a microbial biofilm that is irreversibly attached to the wound matrix. Critical Issues: Using molecular techniques requires a financial outlay for high-cost equipment. They are still too time-consuming to perform and reporting is too delayed for them to be used in routine practice. Finally, they do not differentiate live from dead or pathogenic from nonpathogenic microorganisms. Future Directions: Molecular tools have better documented the composition and organization of the skin flora. Further advances are required to elucidate which among the many bacteria in the DFU flora are likely to be pathogens, rather than colonizers

Staphylococcus aureus Toxins and Diabetic Foot Ulcers: Role in Pathogenesis and Interest in Diagnosis

Infection of foot ulcers is a common, often severe and costly complication in diabetes. Diabetic foot infections (DFI) are mainly polymicrobial, and Staphylococcus aureus is the most frequent pathogen isolated. The numerous virulence factors and toxins produced by S. aureus during an infection are well characterized. However, some particular features could be observed in DFI. The aim of this review is to describe the role of S. aureus in DFI and the implication of its toxins in the establishment of the infection. Studies on this issue have helped to distinguish two S. aureus populations in DFI: toxinogenic S. aureus strains (harboring exfoliatin-, EDIN-, PVL- or TSST-encoding genes) and non-toxinogenic strains. Toxinogenic strains are often present in infections with a more severe grade and systemic impact, whereas non-toxinogenic strains seem to remain localized in deep structures and bone involving diabetic foot osteomyelitis. Testing the virulence profile of bacteria seems to be a promising way to predict the behavior of S. aureus in the chronic wounds

Propolis potentiates the effect of cranberry (Vaccinium macrocarpon) against the virulence of uropathogenic Escherichia coli

International audienceUropathogenic Escherichia coli (UPEC), the most prevalent bacteria isolated in urinary tract infections (UTI), is now frequently resistant to antibiotics used to treat this pathology. The antibacterial properties of cranberry and propolis could reduce the frequency of UTIs and thus the use of antibiotics, helping in the fight against the emergence of antibiotic resistance. Transcriptomic profiles of a clinical UPEC strain exposed to cranberry proanthocyanidins alone (190 \textmug/mL), propolis alone (102.4 \textmug/mL) and a combination of both were determined. Cranberry alone, but more so cranberry + propolis combined, modified the expression of genes involved in different essential pathways: down-expression of genes involved in adhesion, motility, and biofilm formation, and up-regulation of genes involved in iron metabolism and stress response. Phenotypic assays confirmed the decrease of motility (swarming and swimming) and biofilm formation (early formation and formed biofilm). This study showed for the first time that propolis potentiated the effect of cranberry proanthocyanidins on adhesion, motility, biofilm formation, iron metabolism and stress response of UPEC. Cranberry + propolis treatment could represent an interesting new strategy to prevent recurrent UTI

Comparison of Stir Bar Sorptive Extraction and Solid Phase Microextraction of Volatile and Semi-Volatile Metabolite Profile of Staphylococcus Aureus

International audienceFor the analysis of volatile bacterial compounds, solid phase microextraction (SPME) is currently the most widely used metabolite concentration technique. Recently, the potential of stir bar sorptive extraction (SBSE) for this use has been demonstrated. These two approaches were therefore used in combination with gas-chromatography coupled with mass-spectrometry (GC-MS) for the analysis of volatile and semi-volatile bacterial compounds produced by Staphylococcus aureus. In both cases, SPME and SBSE/headspace sorptive extraction (HSSE) enrichment was carried out in two coating phases. A whole analytical and statistical process was developed to differentiate the metabolites produced from the metabolites consumed. The results obtained with SBSE/HSSE and SPME were compared and showed the recovery of 90% of the compounds by SBSE/HSSE. In addition, we were able to detect the production of 12 volatile/semi-volatile compounds by S. aureus, six of which had never been reported before. The extraction by SBSE/HSSE showed higher concentration capacities and greater sensitivity than SPME concerning bacterial compounds, suggesting that this technique may therefore become the new preferred option for bacterial volatile and semi-volatile compound analysis

Editorial: Microbial Biofilms in Chronic and Recurrent Infections

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