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Abstract In functional near infrared spectroscopy (fNIRS), deconvolution analysis of oxy and deoxy-hemoglobin concentration changes allows estimating specific hemodynamic response functions (HRF) elicited by neuronal activity, taking advantage of the fNIRS excellent temporal resolution. Diffuse optical tomography (DOT) is also becoming the new standard reconstruction procedure as it is more accurate than the modified Beer Lambert law approach at the sensor level. The objective of this study was to assess the relevance of HRF deconvolution after DOT constrained along the cortical surface. We used local personalized fNIRS montages which consists in optimizing the position of fNIRS optodes to ensure maximal sensitivity to subject specific target brain regions. We carefully evaluated the accuracy of deconvolution when applied after DOT, using realistic simulations involving several HRF models at different signal to noise ratio (SNR) levels and on real data related to motor and visual tasks in healthy subjects and from spontaneous pathological activity in one patient with epilepsy. We demonstrated that DOT followed by deconvolution was able to accurately recover a large variability of HRFs over a large range of SNRs. We found good performances of deconvolution analysis for SNR levels usually encountered in our applications and we were able to reconstruct accurately the temporal dynamics of HRFs in real conditions.

Abstract Papaver genus, commonly known as popies, is a valuable source of alkaloids used in medicine, including papaverine, morphine, codeine, and thebaine. We isolated six endophytic fungal isolates producing morphinan alkaloids from four Papaver species growing in Kurdistan Province, Iran. To do this, a 1:1 mixture of methanol and chloroform was used to extract fungal cultures. The contents of morphinan alkaloids in the extracts were subsequently determined using phase high-performance liquid chromatography. Among the morphinan alkaloid-producing fungal isolates, IRAN 4653C had the highest yield giving 23.06 (mg/g) morphine and 2.03 (mg/g) codeine when grown in potato dextrose liquid medium. The identity of this isolate was examined and recognized as a new fungal species named as Pithoascus kurdistanesis sp. nov. based on multi-gene phylogenetic analyses of ITS, TEF-1α, and TUB2 sequences data and morphological features. The morphinan-producing endophytic fungus and the isolated Pithoascus species from Papaver are being reported for the first time. Accordingly, this fungus shows promise as a new source of valuable compounds which is illustrated and introduced here as a new Microascaceae member belonging to Pithoascus from Kurdistan Province, Iran. Moreover, the morphinan productivity of P. kurdistanesis was further validated by gas chromatography-mass spectrometry (GC–MS).

Candida auris has emerged as a significant healthcare-associated pathogen due to its multidrug-resistant nature. Ongoing constraints in the discovery and provision of new antifungals create an urgent imperative to design effective remedies to this pressing global blight. Herein, we screened a chemical library and identified aryl-carbohydrazide analogs with potent activity against both C. auris and the most prevalent human fungal pathogen, C. albicans. SPB00525 [N’-(2,6-dichlorophenyl)-5-nitro-furan-2-carbohydrazide] exhibited potent activity against different strains that were resistant to standard antifungals. Using drug-induced haploinsufficient profiling, transcriptomics and metabolomic analysis, we uncovered that Ole1, a Δ(9) fatty acid desaturase, is the likely target of SPB00525. An analog of the latter, HTS06170 [N’-(2,6-dichlorophenyl)-4-methyl-1,2,3-thiadiazole-5-carbohydrazide], had a superior antifungal activity against both C. auris and C. albicans. Both SPB00525 and HTS06170 act as antivirulence agents and inhibited the invasive hyphal growth and biofilm formation of C. albicans. SPB00525 and HTS06170 attenuated fungal damage to human enterocytes and ameliorate the survival of Galleria mellonella larvae used as systemic candidiasis model. These data suggest that inhibiting fungal Δ(9) fatty acid desaturase activity represents a potential therapeutic approach for treating fungal infection caused by the superbug C. auris and the most prevalent human fungal pathogen, C. albicans.

Plasmids carrying antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen. In this study, we describe three new plasmids found in strains of A. salmonicida subsp. salmonicida from Québec, Canada: pAsa-2358, pAsa-2900, and pAsa-2900b. The last two plasmids have a tet(D) resistance gene against tetracycline, which has not been previously found in A. salmonicida. The only difference between these two plasmids is the presence of a florfenicol resistance gene (floR) surrounded by two copies of the same insertion sequence, indicating that pAsa-2900 and pAsa-2900b are plasmid variants. All three plasmids represent an issue for aquaculture because they bear resistance genes against antibiotics used to treat infected fish: floR and tet(A) for pAsa-2358; floR, sul2, and tet(D) for pAsa-2900; and sul2 and tet(D) for pAsa-2900b. Conjugation assays confirmed that the three plasmids are capable of conjugation with one A. salmonicida mesophilic strain, one A. hydrophila strain, and Escherichia coli DH5α; except for pAsa-2358 that is not able to conjugate into E. coli DH5α. Based on the results of antibiotic resistance assays, the tet(D) gene provides a greater resistance to tetracycline than the tet(A) gene found in many A. salmonicida subsp. salmonicida plasmids. This is the case whether the plasmids are in their parental A. salmonicida subsp. salmonicida strains or in Aeromonas sp. transconjugates. The presence of these three plasmids in A. salmonicida subsp. salmonicida strengthens the role of this bacterium as a reservoir for antibiotic resistance genes.

Objectives: This project studied the impact of a chlortetracycline treatment in weaning piglets on the taxonomy and antibiotic resistance gene (ARG) content of the microbiomes on carcasses and loins. Methods: Two groups of piglets from two farrowing barns with either an average or a lower sanitary health status were used. Each group was divided in half: a control group and a treatment group receiving feed supplemented with 660 g of chlortetracycline per tonne for 21 days. The piglets then went through fattening and were sent to the abattoir when they reached the targeted slaughter weight. Results: The microbiomes of the pig carcasses and loins were sampled, and DNA was extracted and sequenced with a whole-genome approach. The microbiomes of the carcasses differed depending on the farrowing barn source in both taxonomical composition and ARG content; however, the microbiomes on the loins were similar, regardless of the farrowing barn source and the treatment group. Conclusions: While there were differences in the carcass microbiomes between treatments after processing by the abattoir, the loin microbiomes were consistent and unaffected by treatment with chlortetracycline or the sanitary status of the farrowing barn.

BackgroundStereotactic body radiotherapy (SBRT) is becoming increasingly used in treating localized prostate cancer (PCa), with evidence showing similar toxicity and efficacy profiles when compared with longer courses of definitive radiation. Magnetic resonance imaging (MRI)-guided radiotherapy has multiple potential advantages over standard computed tomography (CT)-guided radiotherapy, including enhanced prostate visualization (abrogating the need for fiducials and MRI fusion), enhanced identification of the urethra, the ability to track the prostate in real-time, and the capacity to perform online adaptive planning. However, it is unknown whether these potential advantages translate into improved outcomes. This phase III randomized superiority trial is designed to prospectively evaluate whether toxicity is lower after MRI-guided versus CT-guided SBRT.MethodsThree hundred men with localized PCa will be randomized in a 1:1 ratio to SBRT using CT or MRI guidance. Randomization will be stratified by baseline International Prostate Symptom Score (IPSS) (≤15 or > 15) and prostate gland volume (≤50 cc or > 50 cc). Five fractions of 8 Gy will be delivered to the prostate over the course of fourteen days, with or without hormonal therapy and elective nodal radiotherapy (to a dose of 5 Gy per fraction) as per the investigator's discretion. The primary endpoint is the incidence of physician-reported acute grade ≥ 2 genitourinary (GU) toxicity (during the first 90 days after SBRT), as assessed by the CTCAE version 4.03 scale. Secondary clinical endpoints include incidence of acute grade ≥ 2 gastrointestinal (GI) toxicity, 5-year cumulative incidences of physician-reported late grade ≥ 2 GU and GI toxicity, temporal changes in patient-reported quality of life (QOL) outcomes, 5-year biochemical recurrence-free survival and the proportion of fractions of MRI-guided SBRT in which online adaptive radiotherapy is used.DiscussionThe MIRAGE trial is the first randomized trial comparing MRI-guided with standard CT-guided SBRT for localized PCa. The primary hypothesis is that MRI-guided SBRT will lead to an improvement in the cumulative incidence of acute grade ≥ 2 GU toxicity when compared to CT-guided SBRT. The pragmatic superiority design focused on an acute toxicity endpoint will allow an early comparison of the two technologies.Trial registrationClinicaltrials.gov identifier: NCT04384770. Date of registration: May 12, 2020. https://clinicaltrials.gov/ct2/show/NCT04384770 PROTOCOL VERSION: Version 2.1, Aug 28, 2020.

Introduction: To consider the growing health issues caused by antibiotic resistance from a “one health” perspective, the contribution of meat production needs to be addressed. While antibiotic resistance is naturally present in microbial communities, the treatment of farm animals with antibiotics causes an increase in antibiotic resistance genes (ARG) in the gut microbiome. Pigs are among the most prevalent animals in agriculture; therefore, reducing the prevalence of antibiotic-resistant bacteria in the pig gut microbiome could reduce the spread of antibiotic resistance. Probiotics are often studied as a way to modulate the microbiome and are, therefore, an interesting way to potentially decrease antibiotic resistance.Methods: To assess the efficacy of a probiotic to reduce the prevalence of ARGs in the pig microbiome, six pigs received either treatment with antibiotics (tylvalosin), probiotics (Pediococcus acidilactici MA18/5M; Biopower® PA), or a combination of both. Their faeces and ileal digesta were collected and DNA was extracted for whole genome shotgun sequencing. The reads were compared with taxonomy and ARG databases to identify the taxa and resistance genes in the samples.Results: The results showed that the ARG profiles in the faeces of the antibiotic and combination treatments were similar, and both were different from the profiles of the probiotic treatment (p < 0.05). The effects of the treatments were different in the digesta and faeces. Many macrolide resistance genes were detected in a higher proportion in the microbiome of the pigs treated with antibiotics or the combination of probiotics and antibiotics. Resistance-carrying conjugative plasmids and horizontal transfer genes were also amplified in faeces samples for the antibiotic and combined treatments. There was no effect of treatment on the short chain fatty acid content in the digesta or the faeces.Conclusion: There is no positive effect of adding probiotics to an antibiotic treatment when these treatments are administered simultaneously.

ABSTRACTDue to the scarcity of transition metals within the human host, fungal pathogens have evolved sophisticated mechanisms to uptake and utilize these micronutrients at the infection interface. While considerable attention was turned to iron and copper acquisition mechanisms and their importance in fungal fitness, less was done regarding either the role of manganese (Mn) in infectious processes or the cellular mechanism by which fungal cells achieve their Mn-homeostasis. Here, we undertook transcriptional profiling in the pathogenic fungus Candida albicans experiencing both Mn starvation and excess to capture biological processes that are modulated by this metal. We uncovered that Mn scarcity influences diverse processes associated with fungal fitness including invasion of host cells and antifungal sensitivity. We show that Mn levels influence the abundance of iron and zinc emphasizing the complex crosstalk between metals. The deletion of SMF12, a member of Mn Nramp transporters, confirmed its contribution to Mn uptake. smf12 was unable to form hyphae and damage host cells and exhibited sensitivity to azoles. We found that the unfolded protein response (UPR), likely activated by decreased glycosylation under Mn limitation, was required to recover growth when cells were shifted from an Mn-starved to an Mn-repleted medium. RNA-seq profiling of cells exposed to Mn excess revealed that UPR was also activated. Furthermore, the UPR signaling axis Ire1-Hac1 was required to bypass Mn toxicity. Collectively, this study underscores the importance of Mn homeostasis in fungal virulence and comprehensively provides a portrait of biological functions that are modulated by Mn in a fungal pathogen.IMPORTANCETransition metals such as manganese provide considerable functionality across biological systems as they are used as cofactors for many catalytic enzymes. The availability of manganese is very limited inside the human body. Consequently, pathogenic microbes have evolved sophisticated mechanisms to uptake this micronutrient inside the human host to sustain their growth and cause infections. Here, we undertook a comprehensive approach to understand how manganese availability impacts the biology of the prevalent fungal pathogen, Candida albicans. We uncovered that manganese homeostasis in this pathogen modulates different biological processes that are essential for host infection which underscores the value of targeting fungal manganese homeostasis for potential antifungal therapeutics development.

For the precise measurements of special nuclear materials (SNM) including Pu and Am isotopes, we have used phoswich detector combination of two single crystal scintillators of Gd3Ga3Al2O12:Ce and CsI:Tl. High detection efficiency and sensitivity along with high figure of merit for the discrimination of these phoswich detectors ensures the detection and discrimination of thermal neutrons and gammas from spontaneous fission of Pu and other isotopes in presence of high gamma background. Using this detector, the low energy gammas, which is stopped completely in 1mm thick disc of GGAG, can be also discriminated from high energies gamma and shows linearity in wide range of sample quantities. By changing only the appropriate shielding, the similar setup was used for thermal neutron detection and shows a very good linearity over wide range. The quantity of a test sample was also calculated accurately by using the measured calibrated plot.

Abstract Background The quorum-sensing molecule farnesol, in opportunistic yeast Candida albicans, modulates its dimorphic switch between yeast and hyphal forms, and biofilm formation. Although there is an increasing interest in farnesol as a potential antifungal drug, the molecular mechanism by which C. albicans responds to this molecule is still not fully understood. Results A comparative genomic analysis between C. albicans strains that are naturally unresponsive to 30 µM of farnesol on TYE plates at 37 °C versus responsive strains uncovered new molecular determinants involved in the response to farnesol. While no signature gene was identified, amino acid changes in specific proteins were shown to correlate with the unresponsiveness to farnesol, particularly with substitutions in proteins known to be involved in the farnesol response. Although amino acid changes occur primarily in disordered regions of proteins, some amino acid changes were also found in known domains. Finally, the genomic investigation of intermediate-response strains showed that the non-response to farnesol occurs gradually following the successive accumulation of amino acid changes at specific positions. Conclusion It is known that large genomic changes, such as recombinations and gene flow (losses and gains), can cause major phenotypic changes in pathogens. However, it is still not well known or documented how more subtle changes, such as amino acid substitutions, play a role in the adaptation of pathogens. The present study shows that amino acid changes can modulate C. albicans yeast’s response to farnesol. This study also improves our understanding of the network of proteins involved in the response to farnesol, and of the involvement of amino acid substitutions in cellular behavior.

ABSTRACT Transcription factors (TFs) play key roles in cellular regulation and are critical in the control of drug resistance in the fungal pathogen Candida albicans. We found that activation of the transcription factor C4_02500C_A (Adr1) conferred significant resistance against fluconazole. In Saccharomyces cerevisiae, Adr1 is a carbon-source-responsive zinc-finger transcription factor required for transcription of the glucose-repressed gene ADH1 and of genes required for ethanol, glycerol, and fatty acid utilization. Motif scanning of promoter elements suggests that Adr1 may be rewired in fungi and govern the ergosterol synthesis pathway in C. albicans. Because previous studies have identified the zinc-cluster transcription factor Upc2 as a regulator of the ergosterol pathway in both fungi, we examined the relationship between Adr1 and Upc2 in sterol biosynthesis in C. albicans. Phenotypic profiles of either ADR1 or UPC2 modulation in C. albicans showed differential growth in the presence of fluconazole; either adr1 or upc2 homozygous deletion results in sensitivity to the drug, while their activation generates a fluconazole-resistant strain. The rewiring from ergosterol synthesis to fatty acid metabolism involved all members of the Adr1 regulon except the alcohol dehydrogenase Adh1, which remains under Adr1 control in both circuits and may have been driven by the lifestyle of S. cerevisiae, which requires the ability to both tolerate and process high concentrations of ethanol. Importance Research often relies on well-studied orthologs within related species, with researchers using a well-studied gene or protein to allow prediction of the function of the ortholog. In the opportunistic pathogen Candida albicans, orthologs are usually compared with Saccharomyces cerevisiae, and this approach has been very fruitful. Many transcription factors (TFs) do similar jobs in the two species, but many do not, and typically changes in function are driven not by modifications in the structures of the TFs themselves but in the connections between the transcription factors and their regulated genes. This strategy of changing TF function has been termed transcription factor rewiring. In this study, we specifically looked for rewired transcription factors, or Candida-specific TFs, that might play a role in drug resistance. We investigated 30 transcription factors that were potentially rewired or were specific to the Candida clade. We found that the Adr1 transcription factor conferred resistance to drugs like fluconazole, amphotericin B, and terbinafine when activated. Adr1 is known for fatty acid and glycerol utilization in Saccharomyces, but our study reveals that it has been rewired and is connected to ergosterol biosynthesis in Candida albicans.

Introduction: A thorough understanding of the microbial ecology within the swine value chain is essential to develop new strategies to optimize the microbiological quality of pork products. To our knowledge, no study to date has followed the microbiota through the value chain from live farm animals to the cuts of meat obtained for market. The objective of this study is to evaluate how the microbiota of pigs and their environment influence the microbial composition of samples collected throughout the value chain, including the meat plant and meat cuts.Method and results: Results from 16S rDNA sequencing, short-chain fatty acid concentrations and metabolomic analysis of pig feces revealed that the microbiota from two farms with differing sanitary statuses were distinctive. The total aerobic mesophilic bacteria and Enterobacteriaceae counts from samples collected at the meat plant after the pre-operation cleaning and disinfection steps were at or around the detection limit and the pigs from the selected farms were the first to be slaughtered on each shipment days. The bacterial counts of individual samples collected at the meat plant did not vary significantly between the farms. Alpha diversity results indicate that as we move through the steps in the value chain, there is a clear reduction in the diversity of the microbiota. A beta diversity analysis revealed a more distinct microbiota at the farms compared to the meat plant which change and became more uniform as samples were taken towards the end of the value chain. The source tracker analysis showed that only 12.92% of the microbiota in shoulder samples originated from the farms and 81% of the bacteria detected on the dressed carcasses were of unknown origin.Discussion: Overall, the results suggest that with the current level of microbial control at farms, it is possible to obtain pork products with similar microbiological quality from different farms. However, broader studies are required to determine the impact of the sanitary status of the herd on the final products.

Vibrio cholerae includes strains responsible for the cholera disease and is a natural inhabitant of aquatic environments. V. cholerae possesses a unique polar flagellum essential for motility, adhesion, and biofilm formation. In a previous study, we showed that motility and biofilm formation are altered in the presence of subinhibitory concentrations of polymyxin B in V. cholerae O1 and O139. In this study, we performed an experimental evolution to identify the genes restoring the motility in the presence of a subinhibitory concentration of polymyxin B. Mutations in five genes have been identified in three variants derived from two different parental strains A1552 and MO10: ihfA that encodes a subunit of the integration host factor (IHF), vacJ (mlaA) and mlaF, two genes belonging to the maintenance of the lipid asymmetry (Mla) pathway, dacB that encodes a penicillin-binding protein (PBP4) and involved in cell wall synthesis, and ccmH that encodes a c-type cytochrome maturation protein. We further demonstrated that the variants derived from MO10 containing mutations in vacJ, mlaF, and dacB secrete more and larger membrane vesicles that titer the polymyxin B, which increases the bacterial survival and is expected to limit its impact on the bacterial envelope and participate in the flagellum’s retention and motility.

This study was conducted to characterise phenotypically helmeted Guinea fowls in three agro-ecologies in Nigeria using multivariate approach. Eighteen biometric characters, four morphological indices and eleven qualitative physical traits were investigated in a total of 569 adult birds (158 males and 411 females). Descriptive statistics, non-parametric Kruskal-Wallis H test followed by the Mann-Whitney U and Dunn-Bonferroni tests for post hoc, Multiple Correspondence Analysis (MCA), Univariate Analysis, Canonical Discriminant Analysis, Categorical Principal Component Analysis and Decision Trees were employed to discern the effects of agro-ecological zone and sex on the morphostructural parameters. Agro-ecology had significant effect (P

Studies on the microbiome of different species are on the rise, due to a growing interest in animal health and the safety of food products of animal origin. A challenge with studying animals’ microbiomes is to find methods that obtain a good representation of the microbial community of interest. Good unbiased sampling protocols are the basis for a solid experimental design, but may need to be done in environments where sample preservation could be difficult. In this study, we evaluate by shotgun sequencing the impact of stabilizing swine faeces samples using a commercial stabilizer (PERFORMAbiome • GUT | PB-200, DNA Genotek). Using stabilizer makes it possible to obtain DNA that is significantly less degraded than when the samples are not stabilized. Also, the results on the taxonomy and on the bacterial functions encoded in the microbiome are impacted by whether or not the samples are stabilized. Finally, the stabilization of samples that had already been frozen and stored at -80°C led to extraction and DNA quality results similar to those obtained from samples that were stabilized before freezing.

Plasmids that carry antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen with severe consequences in salmonid farming. Here, we describe a 67 kb plasmid found in the A. salmonicida subsp. salmonicida Strain SHY15-2939 from Quebec, Canada. This new plasmid, named pAsa-2939 and identified by high throughput sequencing, displays features never found before in this bacterial species. It contains a transposon related to the Tn21 family, but with an unusual organization. This transposon bears a catB3 gene (chloramphenicol resistance) that has not been detected yet in A. salmonicida subsp. salmonicida. The plasmid is transferable by conjugation into Aeromonas hydrophila, but not into Escherichia coli. Based on PCR analysis and genomic sequencing (Illumina and PacBio), we determined that the transposon is unstable in A. salmonicida subsp. salmonicida Strain SHY15-2939, but it is stable in A. hydrophila trans-conjugants, which explains the chloramphenicol resistance variability observed in SHY15-2939. These results suggest that this bacterium is likely not the most appropriate host for this plasmid. The presence of pAsa-2939 in A. salmonicida subsp. salmonicida also strengthens the reservoir role of this bacterium for antibiotic resistance genes, even those that resist antibiotics not used in aquaculture in Québec, such as chloramphenicol.

Antibiotic resistance is an issue in many areas of human activity. The mobilization of antibiotic resistance genes within the bacterial community makes it difficult to study and control the phenomenon. It is known that certain insertion sequences, which are mobile genetic elements, can participate in the mobilization of antibiotic resistance genes and in the expression of these genes. However, the magnitude of the contribution of insertion sequences to the mobility of antibiotic resistance genes remains understudied. In this study, the relationships between insertion sequences and antibiotic resistance genes present in the microbiome were investigated using two public datasets. The first made it possible to analyze the effects of different antibiotics in a controlled mouse model. The second dataset came from a study of the differences between conventional and organic-raised cattle. Although it was possible to find statistically significant correlations between the insertion sequences and antibiotic resistance genes in both datasets, several challenges remain to better understand the contribution of insertion sequences to the motility of antibiotic resistance genes. Obtaining more complete and less fragmented metagenomes with long-read sequencing technologies could make it possible to understand the mechanisms favoring horizontal transfers within the microbiome with greater precision.

We sampled the first mitogenomes from a novel lineage of the Chlorophyceae that is sister to Sphaeropleales. Although the Jenufa perforata (27,180 bp) and Jenufa minuta (41,488 bp) mitogenomes differ markedly in size, gene order and intron content, their repertoires of conserved genes are essentially identical. Their rRNA genes display the same fragmentation patterns as their counterparts in Sphaeropleales. Their tRNA gene complements include two ancestral genes shared with Oedogoniales and Chaetophorales (Chlorophyceae) as well as a derived gene for a tRNA species decoding UGA as tryptophan. This noncanonical genetic code differs from those previously reported for Sphaeropleales.

We describe the plastome of Jenufa minuta, a unicellular green alga whose precise relationship with the Sphaeropleales (Chlorophyceae) remains uncertain. This 206,680-bp genome contains 100 conserved genes, 11 of which exhibit 16 group I and seven group II introns. We discovered a trans-spliced group II intron in the rpl32 gene of J. minuta and its orthologs in all Sphaeropleales and several Chlamydomonadales; this is the first time this intron is documented. As in the mitochondria of J. minuta, the UGA codon is decoded as tryptophan. To our knowledge, this is the second report of a variant genetic code in plastids.

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) – for comparative purposes in individuals with another chronic neurodegenerative condition – and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD.

Aeromonas salmonicida subsp. salmonicida is a pathogenic bacterium responsible for furunculosis in salmonids. Following an outbreak of furunculosis, the infection can be treated with antibiotics, but it is common to observe ineffective treatment due to antibiotic resistance. This bacterium has a wide variety of plasmids responsible for this resistance. Among them, pRAS3 carries a tetracycline resistance gene. Several variants of this plasmid have been discovered over the years (pRAS3-3432 and pRAS3.1 to 3.4). During the present study, two new variants of the plasmid pRAS3 were identified (pRAS3.5 and pRAS3-3759) in strains of A. salmonicida subsp. salmonicida. Plasmid pRAS3-3759, which has been found in many strains from the same region over the past three years, has an additional genetic element identical to one found in pRAS3-3432. This genetic element was also found in Chlamydia suis, a swine pathogen. In this study, we analyzed the bacteria’s resistance to tetracycline, the number of copies of the plasmids, and the growth of the strains that carry five of the pRAS3 variants (pRAS3.3 to 3.5, pRAS3-3432, and pRAS3-3759). The results show no particular trend despite the differences between the plasmids, except for the resistance to tetracycline when analyzed in an isogenic background. Blast analysis also revealed the presence of pRAS3 plasmids in other bacterial species, which suggests that this plasmid family has widely spread. This study once again highlights the ability of A. salmonicida subsp. salmonicida to adapt to furunculosis antibiotic treatments, and the still-growing family of pRAS3 plasmids.

It has been previously shown that RskA, the anti-Sigma factor K of Mycobacterium tuberculosis, inhibits SigK and that mutations in RskA promote high expression of the SigK regulon. The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function. In this report, we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership. Consistent with predictions, the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK. Unexpectedly, RskA also served as an activator of SigK. This activation depended on the same N-terminal region and was enhanced by the membrane-extracellular portion of RskA. Based on this, we engineered similar truncations in a Gram-negative bacterium, namely Yersinia enterocolitica. Again, we observed that, with specific alterations of RskA, we were able to enhance SigK activity. Together these results support an alternative mechanism of anti-Sigma factor function, that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria, suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized.

Abstract Objective Pseudomonas aeruginosa is an opportunistic bacterial pathogen well known to cause chronic lung infections in individuals with cystic fibrosis (CF). Some strains adapted to this particular niche show distinct phenotypes, such as biofilm hyperproduction. It is necessary to study CF clinical P. aeruginosa isolates, such as Liverpool Epidemic Strains (LES), to acquire a better understanding of the key genes essential for in vivo maintenance and the major virulence mechanisms involved in CF lung infections. Previously, a library of 9216 mutants of the LESB58 strain were generated by signature-tagged mutagenesis (STM) and screened in the rat model of chronic lung infection, allowing the identification of 163 STM mutants showing defects in in vivo maintenance. Results In the present study, these 163 mutants were successively screened in two additional surrogate host models (the amoeba and the fruit fly). The STM PALES_11731 mutant was the unique non-virulent in the three hosts. A competitive index study in rat lungs confirmed that the mutant was 20-fold less virulent than the wild-type strain. This study demonstrated the pertinence to use a multi-host approach to study the genetic determinants of P. aeruginosa strains infecting CF patients.

The bacterium Aeromonas salmonicida has long been known to be one of the most feared pathogens in fish farming. However, the more we discover about this bacterial species, the more we question whether it is really exclusively an aquatic pathogen. In recent years, it has become obvious that this bacterial species includes a myriad of strains with various lifestyle and ecological niches, including the well-known strict psychrophiles, the first bacteria known of the species, and the newly described mesophilic strains. The mesophiles are able to grow at low temperatures, but even better at temperatures of approximately 37 °C, which strict psychrophiles cannot do. In this perspective article, we address some aspects surrounding this dual lifestyle in A. salmonicida, including the impact of mobile genetic elements, and how future research around this bacterial species may focus on the psychrophilic/mesophilic dichotomy, which makes A. salmonicida an increasingly interesting and relevant model for the study of speciation.

Pork meat is in high demand worldwide and this is expected to increase. Pork is often raised in intensive conditions, which is conducive to the spread of infectious diseases. Vaccines, antibiotics, and other biosafety measures help mitigate the impact of infectious diseases. However, bacterial strains resistant to antibiotics are more and more frequently found in pig farms, animals, and the environment. It is now recognized that a holistic perspective is needed to sustainably fight antibiotic resistance, and that an integrated One Health approach is essential. With this in mind, this review tackles antibiotic resistance throughout the pork raising process, including their microbiome; many factors of their environment (agricultural workers, farms, rivers, etc.); and an overview of the impact of antibiotic resistance on pork meat, which is the end product available to consumers. Antibiotic resistance, while a natural process, is a public health concern. If we react, and act, collectively, it is expected to be, at least partially, reversible with judicious antibiotic usage and the development of innovative strategies and tools to foster animal health.

Abstract Phages infecting Aeromonas salmonicida subsp. salmonicida, the causative agent of the fish disease furunculosis, have been isolated for decades but very few of them have been characterized. Here, the host range of 12 virulent phages, including three isolated in the present study, was evaluated against a panel of 65 A. salmonicida isolates, including representatives of the psychrophilic subspecies salmonicida, smithia, masoucida, and the mesophilic subspecies pectinolytica. This bacterial set also included three isolates from India suspected of being members of a new subspecies. Our results allowed to elucidate a lytic dichotomy based on the lifestyle of A. salmonicida (mesophilic or psychrophilic) and more generally, on phage types (lysotypes) for the subspecies salmonicida. The genomic analyses of the 12 phages from this study with those available in GenBank led us to propose an A. salmonicida phage pan-virome. Our comparative genomic analyses also suggest that some phage genes were under positive selection and A. salmonicida phage genomes having a discrepancy in GC% compared to the host genome encode tRNA genes to likely overpass the bias in codon usage. Finally, we propose a new classification scheme for A. salmonicida phages.

Abstract Background Aeromonas salmonicida subsp. salmonicida is a ubiquitous psychrophilic waterborne bacterium and a fish pathogen. The numerous mobile elements, especially insertion sequences (IS), in its genome promote rearrangements that impact its phenotype. One of the main virulence factors of this bacterium, its type three secretion system (TTSS), is affected by these rearrangements. In Aeromonas salmonicida subsp. salmonicida most of the TTSS genes are encoded in a single locus on a large plasmid called pAsa5, and may be lost when the bacterium is cultivated at a higher temperature (25 °C), producing non-virulent mutants. In a previous study, pAsa5-rearranged strains that lacked the TTSS locus on pAsa5 were produced using parental strains, including 01-B526. Some of the generated deletions were explained by homologous recombination between ISs found on pAsa5, whereas the others remained unresolved. To investigate those rearrangements, short- and long-read high-throughput sequencing technologies were used on the A. salmonicida subsp. salmonicida 01-B526 whole genome. Results Whole genome sequencing of the 01-B526 strain revealed that its pAsa5 has an additional IS copy, an ISAS5, compared to the reference strain (A449) sequence, which allowed for a previously unknown rearrangement to occur. It also appeared that 01-B526 bears a second large plasmid, named pAsa9, which shares 40 kbp of highly similar sequences with pAsa5. Following these discoveries, previously unexplained deletions were elucidated by genotyping. Furthermore, in one of the derived strains a fusion of pAsa5 and pAsa9, involving the newly discovered ISAS5 copy, was observed. Conclusion The loss of TTSS and hence virulence is explained by one consistent mechanism: IS-driven homologous recombination. The similarities between pAsa9 and pAsa5 also provide another example of genetic diversity driven by ISs.

The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species.