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Abstract Background Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF. Results We demonstrated that HQNO stimulates S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the α-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background. Conclusions These results suggest that S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.

Staphylococcus aureus intramammary infections often leads to clinical and subclinical mastitis in dairy cattle. Prediction of disease evolution and treatment efficacy based on the characteristics of disease-causing strains of S. aureus would significantly improve management of dairy herds. To study the impact of biofilm production and the influence of genetic lineage, we selected S. aureus isolates from the most prevalent Canadian spa types associated with bovine mastitis. Antimicrobial susceptibility in planktonic growth and for bacteria embedded in biofilm was compared. PCR was used to detect the bap gene responsible for atypical biofilm formation. All Canadian spa types from dairy cattle were susceptible to the 8 antimicrobial agents tested. Only strain sa3493 from spa type t267 showed a resistance to pirlimycin. However, bacteria producing larger amounts of biofilms better survived the bactericidal action of antimicrobial agents even when exposed to concentrations 64 folds higher than the minimal inhibitory concentration determined for planktonic cultures. Pirlimycin was more effective on bacteria producing low to moderate levels of biofilm compared with vancomycin or ceftiofur. Antimicrobial agents did not affect the viability of spa types t13401 and t605 that were high biofilm producers. While both these spa types produced high amounts of biofilm, only t605 possessed the bap gene. We also found a close relationship between DIM at sampling and the presence of spa type t605 isolates. These results suggest that detection of S. aureus spa type may help predict the effectiveness of antimicrobial therapy and that some spa types are more likely to be retrieved toward the end of the lactation., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Tomatidine (TO) is a natural narrow-spectrum antibiotic acting on the Staphylococcus aureus small colony variant (SCV) with a minimal inhibitory concentration (MIC) of 0.06 µg/mL while it shows no activity against prototypical strains (MIC > 128 µg/mL). To expand the spectrum of activity of TO, the 3β-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, TM-02 (C3-β) and TM-03 (C3-α). These molecules are equally potent against prototypical S. aureus and E. coli strains (MIC 8 and 32 µg/mL, respectively), whereas TM-02 is more potent against SCV (MIC 0.5 µg/mL) and hyperpermeable E. coli strains (MIC 1 µg/mL). The differences in their modes of action were investigated. We used membrane vesicles to confirm the inhibition of the bacterial ATP synthase, the documented target of TO, and measured effects on bacterial cell membranes. Both molecules inhibited E. coli ATP synthase, with K i values of 1.1 µM and 3.5 µM for TM-02 and TM-03 , respectively, and the bactericidal effect of TM-02 was linked to ATP synthase inhibition. Furthermore, TM-02 had no major effect on the membrane fluidity and gradually reduced membrane potential. In contrast, TM-03 caused structural damages to membranes and completely disrupted the membrane potential (>90%). We were successful in broadening the spectrum of activity of TO. C3-β-diastereoisomers may have more specific antibacterial action than C3-α.

Klebsiella oxytoca is a gram-negative bacterium found in fecal microbiota and known to cause several infections in humans, including antibiotic-associated hemorrhagic colitis. We present here a case of colitis caused by K. oxytoca toxin-producing strains that evolved in chronic diarrhea successfully treated by fecal microbiota transplant., Competing Interests: Potential conflicts of interest. L.-C. F. reports travel support from the Faculty of Medicine and Health Sciences of the University of Sherbrooke and has issued or pending patents for Clostridium difficile toxins A and/or B antigen and epitope antibody (PCT/CA2016/050170; WO/2016/131157 #62118450). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antibiotic resistance is escalating alarmingly worldwide. Bacterial resistance mechanisms are surfacing and proliferating across the globe, jeopardizing our capacity to manage prevalent infectious illnesses. Without drastic measures, we risk entering a post-antibiotic era, where even trivial infections and injuries can cause death again. In this context, we have developed a new class of antibiotics based on tomatidine (TO), a natural product derived from tomato plants, with a novel mode of action by targeting bacterial ATP synthases. The first generation of compounds proved highly specific for small-colony variants (SCVs) of Staphylococcus aureus. However, optimization of this scaffold through extensive structure-activity relationship studies has enabled us to broaden its effectiveness to include both Gram-positive and Gram-negative bacteria. Notably, the results showed that specific C3-modification of TO could improve ATP synthase inhibition and also bypass the outer membrane barrier of Gram-negative bacteria to gain substantial growth inhibition including against multi-resistant strains., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Pierre-Luc Boudreault reports financial support was provided by CIHR. Pierre-Luc Boudreault has patent pending to Université de Sherbrooke., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Staphylococcus aureus is one of the major pathogens causing bovine mastitis, and antibiotic treatment is most often inefficient due to its virulence and antibiotic-resistance attributes. The development of new antibiotics for veterinary use should account for the One Health concept, in which humans, animals, and environmental wellbeing are all interconnected. S. aureus can infect cattle and humans alike and antibiotic resistance can impact both if the same classes of antibiotics are used. New effective antibiotic classes against S. aureus are thus needed in dairy farms. We previously described PC1 as a novel antibiotic, which binds the S. aureus guanine riboswitch and interrupts transcription of essential GMP synthesis genes. However, chemical instability of PC1 hindered its development, evaluation, and commercialization. Novel PC1 analogs with improved stability have now been rationally designed and synthesized, and their in vitro and in vivo activities have been evaluated. One of these novel compounds, PC206, remains stable in solution and demonstrates specific narrow-spectrum activity against S. aureus . It is active against biofilm-embedded S. aureus , its cytotoxicity profile is adequate, and in vivo tests in mice and cows show that it is effective and well tolerated. PC206 and structural analogs represent a promising new antibiotic class to treat S. aureus -induced bovine mastitis.

Steroidal (glycol)alkaloids S(G)As are secondary metabolites made of a nitrogen-containing steroidal skeleton linked to a (poly)saccharide, naturally occurring in the members of the Solanaceae and Liliaceae plant families. The genus Solanum is familiar to all of us as a food source (tomato, potato, eggplant), but a few populations have also made it part of their ethnobotany for their medicinal properties. The recent development of the isolation, purification and analysis techniques have shed light on the structural diversity among the SGAs family, thus attracting scientists to investigate their various pharmacological properties. This review aims to overview the recent literature (2012-2022) on the pharmacological benefits displayed by the SGAs family. Over 17 different potential therapeutic applications (antibiotic, antiviral, anti-inflammatory, etc.) were reported over the past ten years, and this unique review analyzes each pharmacological effect independently without discrimination of either the SGA's chemical identity or their sources. A strong emphasis is placed on the discovery of their biological targets and the subsequent cellular mechanisms, discussing in vitro to in vivo biological data. The therapeutic value and the challenges of the solanum steroidal glycoalkaloid family is debated to provide new insights for future research towards clinical development.

Prototypic Staphylococcus aureus and their small-colony variants (SCVs) are predominant in cystic fibrosis (CF), but the interdependence of these phenotypes is poorly understood. We characterized S. aureus isolates from adult CF patients over several years. Of 18 S. aureus -positive patients (58%), 13 (72%) were positive for SCVs. Characterization included genotyping, SCCmec types, auxotrophy, biofilm production, antibiotic susceptibilities and tolerance, and resistance acquisition rates. Whole-genome sequencing revealed that several patients were colonized with prototypical and SCV-related clones. Some clonal pairs showed acquisition of aminoglycoside resistance that was not explained by aminoglycoside-modifying enzymes, suggesting a mutation-based process. The characteristics of SCVs that could play a role in resistance acquisition were thus investigated further. For instance, SCV isolates produced more biofilm ( p < 0.05) and showed a higher survival rate upon exposure to ciprofloxacin and vancomycin compared to their prototypic associated clones. SCVs also developed spontaneous rifampicin resistance mutations at a higher frequency. Accordingly, a laboratory-derived SCV (Δ hemB ) acquired resistance to ciprofloxacin and gentamicin faster than its parent counterpart after serial passages in the presence of sub-inhibitory concentrations of antibiotics. These results suggest a role for SCVs in the establishment of persistent antibiotic-resistant clones in adult CF patients.

Staphylococcus aureus causes intramammary infections (IMIs), which are refractory to antibiotic treatment and frequently result in chronic mastitis. IMIs are the leading cause of conventional antibiotic use in dairy farms. Phage therapy represents an alternative to antibiotics to help better manage mastitis in cows, reducing the global spread of resistance. A mouse mastitis model of S. aureus IMI was used to study the efficacy of a new cocktail of five lytic S. aureus -specific phages (StaphLyse™), administered either via the intramammary (IMAM) route or intravenously (IV). The StaphLyse™ phage cocktail was stable in milk for up to one day at 37 °C and up to one week at 4 °C. The phage cocktail was bactericidal in vitro against S. aureus in a dose-dependent manner. A single IMAM injection of this cocktail given 8 h after infection reduced the bacterial load in the mammary glands of lactating mice infected with S. aureus , and as expected, a two-dose regimen was more effective. Prophylactic use (4 h pre-challenge) of the phage cocktail was also effective, reducing S. aureus levels by 4 log10 CFU per gram of mammary gland. These results suggest that phage therapy may be a viable alternative to traditional antibiotics for the control of S. aureus IMIs.

Bacterial biofilms are structured clusters of bacterial cells enclosed in a self-produced polymer matrix that are attached to a biotic or abiotic surface. This structure protects bacteria from hostile environmental conditions. There are also accumulating reports about bacterial aggregates associated but not directly adherent to surfaces. Interestingly, these bacterial aggregates exhibit many of the same phenotypes as surface-attached biofilms. Surface-attached biofilms as well as non-attached aggregates are ubiquitous and found in a wide variety of natural and clinical settings. This strongly suggests that biofilm/aggregate formation is important at some steps in the bacterial lifecycle. Biofilm/aggregate formation might therefore be important for some bacterial species for persistence within their host or their environment, while for other bacterial species it might be more important for persistence in the environment between infection of different individuals or even between infection of different hosts (humans or animals). This is strikingly similar to the One Health concept which recognizes that the health and well-being of humans, animals and the environment are intricately linked. We would like to propose that within this One Health concept, the One Biofilm concept also exists, where biofilm/aggregate formation in humans, animals and the environment are also intricately linked. Biofilm/aggregates could represent the unifying factor underneath the One Health concept. The One Biofilm concept would support that biofilm/aggregate formation might be important for persistence during infection but might as well be even more important for persistence in the environment and for transmission between different individuals/different hosts., (© 2022. The Author(s).)

Non-aureus staphylococci (NAS) and Staphylococcus aureus are pathogens that cause bovine mastitis, a costly disease for dairy farmers, however; many NAS are considered part of the normal udder microbiota. It has been suggested that through a mechanism that remains to be elucidated, NAS intramammary colonization can prevent subsequent infection with other bacterial pathogens. This study shows that in a murine mastitis model, secondary Staph. aureus intramammary colonization is reduced by exoproducts from Staph. chromogenes and Staph. simulans, both NAS, while Streptococcus spp. exoproducts have much less ability to affect the course of the infection caused by S. aureus., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. The versatile and adaptative evolutionary strategies of this bacterium have challenged mastitis control and prevention globally, and the high incidence of S. aureus mastitis increases concerns about antimicrobial resistance (AMR) and zoonosis. This study aims to describe the evolutionary relationship between bovine intramammary infection (IMI)-associated S. aureus and human pathogenic S. aureus and further elucidate the specific genetic composition that leads to the emergence of successful bovine IMI-associated S. aureus lineages. We performed a phylogenomic analysis of 187  S . aureus isolates that originated from either dairy cattle or humans. Our results revealed that bovine IMI-associated S. aureus isolates showed distinct clades compared to human-originated S. aureus isolates. From a pan-genome analysis, 2070 core genes were identified. Host-specific genes and clonal complex (CC)-specific genes were also identified in bovine S. aureus isolates, mostly located in mobile genetic elements (MGEs). Additionally, the genome sequences of three apparent human-adapted isolates (two from CC97 and one from CC8), isolated from bovine mastitis samples, may provide an snapshot of the genomic characteristics in early host spillover events. Virulence and AMR genes were not conserved among bovine IMI-associated S. aureus isolates. Restriction-modification (R-M) genes in bovine IMI-associated S. aureus demonstrated that the Type I R-M system was lineage-specific and Type II R-M system was sequence type (ST)-specific. The distribution of exclusive, virulence, and AMR genes were closely correlated with the presence of R-M systems in S. aureus , suggesting that R-M systems may contribute to shaping clonal diversification by providing a genetic barrier to the horizontal gene transfer (HGT). Our findings indicate that the CC or ST lineage-specific R-M systems may limit genetic exchange between bovine-adapted S. aureus isolates from different lineages.

Antibiotics are the most effective strategy to prevent and treat intramammary infections. However, their misuse has led to the dissemination of multidrug resistant bacteria (MDR) for both animals and humans. Efforts to develop new alternative strategies to control bacterial infections related to MDR are continuously on the rise. The objective of this study was to evaluate the antimicrobial activity of different bacteriocins and reuterin against MDR Staphylococcus and Streptococcus clinical isolates involved in bovine mastitis. A bacterial collection including S. aureus ( n = 19), S. dysgalactiae ( n = 17) and S. uberis ( n = 19) was assembled for this study. Antibiotic resistance profiles were determined by the disk diffusion method. In addition, sensitivity to bacteriocins and reuterin was evaluated by determining minimum inhibitory concentrations (MIC). A total of 21 strains (37.5%) were MDR. MICs ranged from ≤1.0 μg/mL to ≥100 μg/mL for nisin and 2.0 to ≥250 μg/mL for bactofencin. Reuterin was active against all tested bacteria, and MICs vary between 70 and 560 μg/mL. Interestingly, 20 MDR strains were inhibited by bactofencin at a concentration of ≤250 μg/mL, while 14 were inhibited by nisin at an MIC of ≤100 μg/mL. Pediocin did not show an inhibitory effect.

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus., (© 2021, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMI). Standard antibiotic treatments are not very effective and currently available vaccines lack tangible efficacy. Developing a vaccine formulation for S. aureus mastitis is challenging and selection of target antigens is critical. The gene products of six S. aureus genes that are highly expressed during IMI were selected as antigens for this study. The vaccine contained six recombinant proteins formulated with Emulsigen®-D, a CpG oligodeoxynucleotide and indolicidin. Nine cows in mid-lactation received the vaccine while ten received saline (placebo). Two immunizations were performed 10 weeks apart. All the antigens induced an immune response. A balanced immune response (IgG2/IgG1 ratio of 1) was observed for antigen SACOL0442 while a predominant Th2 response was observed for the other antigens (IgG2/IgG1 ratio <1). Immunizations induced CD4+ cell proliferation in response to SACOL0442, SACOL0029, SACOL0720 and SACOL1912 while a CD8+ cell proliferation was induced by SACOL0720. Four weeks after the second immunization, three quarters per animal were experimentally infused with ∼60 CFU of S. aureus. Although no difference in S. aureus counts was observed between the two groups after this robust infectious challenge, a sustained reduction in milk somatic cells counts (SCC) was observed in vaccinated cows. A correlation between SCC and S. aureus counts in milk was also observed. Altogether, this indicates that the collective immune responses induced by the antigens certainly contribute to the observed benefits of the whole vaccine. More work is needed to understand how different antigens stimulate a different response using the same adjuvant., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Motor imagery (MI) is usually facilitated when performed in a congruent body position to the imagined movement, as well as after actual execution (AE). A lower-limb amputation (LLA) results in important structural and functional changes in the sensorimotor system, which can alter MI. In this study, we investigated the effects of body position and AE on the temporal characteristics of MI in people with LLA. Ten participants with LLA (mean age = 59.6 ± 13.9 years, four females) and ten gender- and age-matched healthy control participants (mean age = 60.1 ± 15.4 years, four females) were included. They performed two locomotor-related tasks (a walking task and the Timed Up and Go task) while MI times were measured in different conditions (in congruent/incongruent positions and before/after AE). We showed that MI times were significantly shorter when participants imagined walking in a congruent-standing position compared to an incongruent-sitting position, and when performing MI after actual walking compared to before, in both groups. Shorter MI times in the congruent position and after AE suggest an improvement of MI's temporal accuracy (i.e. the ability to match AE time during MI) in healthy individuals but not in the LLA group.

Listeria monocytogenes ( L. monocytogenes ) is often associated with processed food as it can form biofilms that represent a source of contamination at all stages of the manufacturing chain. The control and prevention of biofilms in food-processing plants are of utmost importance. This study explores the efficacy of prospect molecules for counteracting bacterial mechanisms leading to biofilm formation. The compounds included the phytomolecule tomatidine, zinc chloride (ZnCl 2 ), ethylenediaminetetraacetic acid (EDTA), and a more complexed mixture of bacterial compounds from coagulase-negative staphylococci (CNS exoproducts). Significant inhibition of L. monocytogenes biofilm formation was evidenced using a microfluidic system and confocal microscopic analyses ( p < 0.001). Active molecules were effective at an early stage of biofilm development (≥50% of inhibition) but failed to disperse mature biofilms of L. monocytogenes . According to our findings, prevention of surface attachment was associated with a disruption of bacterial motility. Indeed, agar cell motility assays demonstrated the effectiveness of these molecules. Overall, results highlighted the critical role of motility in biofilm formation and allow to consider flagellum-mediated motility as a promising molecular target in control strategies against L. monocytogenes in food processing environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Doghri, Cherifi, Goetz, Malouin, Jacques and Fravalo.)

Oligomerization of antimicrobial peptides into nanosized supramolecular complexes produced in biological systems (inclusion bodies and self-assembling nanoparticles) seems an appealing alternative to conventional antibiotics. In this work, the antimicrobial peptide, GWH1, was N-terminally fused to two different scaffold proteins, namely, GFP and IFN-γ for its bacterial production in the form of such recombinant protein complexes. Protein self-assembling as regular soluble protein nanoparticles was achieved in the case of GWH1-GFP, while oligomerization into bacterial inclusion bodies was reached in both constructions. Among all these types of therapeutic proteins, protein nanoparticles of GWH1-GFP showed the highest bactericidal effect in an in vitro assay against Escherichia coli , whereas non-oligomerized GWH1-GFP and GWH1-IFN-γ only displayed a moderate bactericidal activity. These results indicate that the biological activity of GWH1 is specifically enhanced in the form of regular multi-display configurations. Those in vitro observations were fully validated against a bacterial infection using a mouse mastitis model, in which the GWH1-GFP soluble nanoparticles were able to effectively reduce bacterial loads.

Siderophores produced in soil by plant growth-promoting rhizobacteria (PGPRs) play several roles, including nutrient mobilizers and can be useful as plants defense elicitors. We investigated the role of a synthetic mixed ligand bis-catechol-mono-hydroxamate siderophore (SID) that mimics the chemical structure of a natural siderophore, fimsbactin, produced by Acinetobacter spp. in the resistance against the phytopathogen Pseudomonas syringae pv tomato DC3000 ( Pst DC3000), in Arabidopsis thaliana. We first tested the antibacterial activity of SID against Pst DC3000 in vitro . After confirming that SID had antibacterial activity against Pst DC3000, we tested whether the observed in vitro activity could translate into resistance of Arabidopsis to Pst DC3000, using bacterial loads as endpoints in a plant infection model. Furthermore, using quantitative polymerase chain reaction, we explored the molecular actors involved in the resistance of Arabidopsis induced by SID. Finally, to assure that SID would not interfere with PGPRs, we tested in vitro the influence of SID on the growth of a reference PGPR, Bacillus subtilis . We report here that SID is an antibacterial agent as well as an inducer of systemic priming of resistance in A. thaliana against Pst DC3000, and that SID can, at the same time, promote growth of a PGPR.

Tomatidine (TO), a steroid alkaloid, exerts a strong bactericidal activity on the infection-persistent phenotype of Staphylococcus aureus , the small-colony variant (SCV), with a minimal inhibitory concentration (MIC) of 0.06 μg/ml. Also, the combination of TO to an aminoglycoside (AMG) shows a strong synergistic effect against prototypical (WT) S. aureus (MIC 0.06 μg/ml), which is otherwise unaffected by TO alone (MIC > 128 μg/ml). We have recently established that the ATP synthase (subunit AtpE) was the molecular target of TO and that TO reduces the production of ATP in S. aureus . The purpose of this study was to understand how TO and the TO-AMG combination exert bactericidal activities against S. aureus SCV and WT strains, respectively. The impact of TO and of the TO-gentamicin (GEN) combination on the membrane potential and generation of reactive oxygen species (ROS) were determined using florescent probes. GEN uptake in WT was assessed in the presence of TO. Virulence of SCV and WT strains as well as of in vitro -selected mutants showing resistance to TO or the TO-GEN combination was evaluated in a murine thigh infection model. TO causes a reduction in membrane potential in both WT and SCV, but significant amounts of ROS are only produced in SCVs. Besides, the presence of TO improves the uptake of GEN by the WT strain and the combination TO-GEN generated 2.5-folds more ROS in WT, compared to that induced by GEN alone. Under anaerobic conditions, WT adopts a fermentative slow-growth phenotype and becomes susceptible to TO even if used alone. In vivo , TO- or TO-GEN-resistant strains were significantly altered in their ability to colonize tissues. These results shed light on the mechanism of action of TO and its synergy with AMGs against S. aureus WT. TO bactericidal activity against SCVs is attributable to both a critical drop in the membrane potential accompanied by a substantial ROS production. In the WT, TO helps GEN uptake and ROS is also important for the synergy. Acquiring resistance to TO significantly impairs virulence. The residual ATP synthase activity of SCVs might represent the Achilles' heel of persistent S. aureus ., (Copyright © 2020 Langlois, Millette, Guay, Dubé-Duquette, Chamberland, Jacques, Rodrigue, Bouarab, Marsault and Malouin.)

Purpose: This study aimed to portray the characteristics, process variables, and sensorimotor outcomes of patients who had received their usual post-stroke in-patient rehabilitation in three stroke rehabilitation units in Quebec in 2013-2014. Method: We assessed patients ( n  = 264) at admission and discharge with a subset of a standardized assessment toolkit consisting of observational and performance-based assessment tools. Results: The patients, with a mean age of 60.3 (SD 15.4) years, were admitted 27.7 (SD 8.4) days post-stroke onset. They had a mean admission FIM score of 83.0 (SD 24.0), a mean length of stay of 48.4 (SD 31.1) days, a mean FIM discharge score of 104.0 (SD 17.0), and a mean FIM efficiency score of 0.44 (SD 0.29). All patient outcomes were significantly improved ( p  < 0.001) and clinically meaningful at discharge (moderate to large Glass's Δ effect sizes) with the improvements greater than or equal to the minimal detectable change at the 95% confidence level in 34%-75% of the patients. Improvements were larger on five of seven outcomes in a sub-group of patients with more severe stroke. Conclusions: The use of a combination of observational and performance assessment tools was essential to capture the full range of disabilities. We have documented significant and clinically meaningful improvements in functional independence, disability, and upper and lower extremity functions after usual post-stroke in-patient rehabilitation in the province of Quebec and provided baseline data for future studies., (© Canadian Physiotherapy Association.)

This study was conducted to estimate the prevalence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) in retail chicken meat and broiler chickens from the Province of Quebec, Canada, and to characterize LA-MRSA isolates. A total of 309 chicken drumsticks and thighs were randomly selected in 2013 from 43 retail stores in the Monteregie. In addition, nasal swabs and caeca samples were collected in 2013-2014 from 200 broiler chickens of 38 different flocks. LA-MRSA was not detected in broiler chickens. Fifteen LA-MRSA isolates were recovered from four (1.3%) of the 309 chicken meat samples. Multi-Locus Sequence Typing (MLST) and SCCmec typing revealed two profiles (ST398-MRSA-V and ST8-MRSA-IVa), which were distinct using pulse-field gel electrophoresis (PFGE) and microarray (antimicrobial resistance and virulence genes) analyses. In addition to beta-lactam resistance, tetracycline and spectinomycin resistance was detected in all isolates from the 3 positive samples of the ST398 profile. Southern blot hybridization revealed that the resistance genes aad(D) and lnu(A), encoding resistances to aminoglycosides and lincosamides respectively, were located on plasmid. All isolates were able to produce biofilms, but biofilm production was not correlated with hld gene expression. Our results show the presence of two separate lineages of MRSA in retail chicken meat in Quebec, one of which is likely of human origin., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus is known to produce persistent and chronic infections in both humans and animals. It is recognized that small-colony variants (SCVs), which produce higher levels of biofilm and that are capable of intracellular persistence, contribute to the chronicity or recurrence of infections and that this phenotype is inherent to the pathogenesis process. Prevention of S. aureus infections through vaccination has not yet met with considerable success. Some of the current vaccine formulations for S. aureus bovine mastitis consist of inactivated S. aureus bacteria, sometimes combined to E. coli J5. As such, the stimulation of cell-mediated immunity by these vaccines might not be optimal. With this in mind, we recently engineered a genetically stable double mutant SCV (ΔvraGΔhemB), which was highly attenuated in a mastitis model of infection. The present work describes the immune responses elicited in mice by various experimental vaccine compositions including the live-attenuated SCV double mutant and its inactivated form, combined or not with inactivated E. coli J5. The live-attenuated SCV was found to provoke a strong and balanced humoral response in immunized mice, as well as strong proliferation of ex-vivo stimulated splenocytes isolated from these animals. These splenocytes were also found to release high concentration of IL-17 and IFN-γ when compared to every other vaccination formulation. Inversely, the inactivated whole-cell vaccine, alone or in combination with the E. coli J5 bacterin, elicited lower antibody titers and failed to induce Th1 or Th17 cell-mediated responses in the splenocyte proliferation assay. Our results suggest that live-attenuated SCVs can trigger host immunity differently than inactivated bacteria and could represent a suitable vector for inducing strong humoral and cell-mediated immune responses, which are crucial for protection. This could represent an important improvement over existing vaccine formulations for preventing S. aureus bovine mastitis and other infections caused by this pathogen., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus and Pseudomonas aeruginosa are prevalent lung pathogens in cystic fibrosis (CF). Whereas co-infection worsens the clinical outcome, prototypical strains are usually antagonistic in vitro . We sought to resolve the discrepancy between these in vitro and in vivo observations. In vitro , growth kinetics for co-cultures of co-isolates from CF patients showed that not all P. aeruginosa strains affected S. aureus viability. On solid media, S. aureus slow-growing colonies were visualized around some P. aeruginosa strains whether or not S. aureus viability was reduced in liquid co-cultures. The S. aureus-P. aeruginosa interactions were then characterized in a mouse lung infection model. Lung homogenates were plated on selective media allowing colony counts of either bacterium. Overall, 35 P. aeruginosa and 10 S. aureus strains (clinical, reference, and mutant strains), for a total of 200 co-infections, were evaluated. We observed that S. aureus colonization of lung tissues was promoted by P. aeruginosa and even by strains showing antagonism in vitro . Promotion was proportional to the extent of P. aeruginosa colonization, but no correlation was found with the degree of myeloperoxidase quantification (as marker of inflammation) or with specific virulence-associated factors using known mutant strains of S. aureus and P. aeruginosa . On the other hand, P. aeruginosa significantly increased the expression of two possible cell receptors for S. aureus , i.e ., ICAM-1 and ITGA-5 (marker for integrin α 5 β 1 ) in lung tissue, while mono-infections by S. aureus did not. This study provides insights on polymicrobial interactions that may influence the progression of CF-associated pulmonary infections., (Copyright © 2019 Millette, Langlois, Brouillette, Frost, Cantin and Malouin.)

Purpose: This study describes the development of a standardized assessment toolkit (SAT) and associated clinical database focusing on sensorimotor rehabilitation in three stroke rehabilitation units (SRUs). Implementation of the SAT was confirmed using objective measures of clinician adherence while exploring reasons for varied adherence. Method: Participants were patients post-stroke admitted for inpatient rehabilitation and clinicians from the three SRUs. A collaborative and iterative process was used to develop the SAT. Implementation was measured by clinician adherence, which was charted by means of assessment entries in patient records and transferred to the clinical database. Reasons for lower adherence were interpreted from therapist data logs at one SRU. Results: The SAT consisted of 25 assessment tools. Clinician adherence to a subset of the tools ranged from 33% to 99% at admission and from 28% to 94% at discharge. At one site, lower adherence among the tools was explained by patient-related factors (1%-36%) and protocol or logistical reasons (0%-7%) at admission; missing data ranged from 0% to 3%, except for the Montreal Cognitive Assessment (17%). Conclusions: In this pragmatic study, objective measures of clinician adherence demonstrated the feasibility of implementing an SAT in daily practice. Moreover, the reasons for lower adherence rates may be related to the patients, protocol, and logistics, all of which may vary with the assessment tool, rather than clinician compliance., Competing Interests: Competing Interests: None declared. This work was supported by the Canadian Institutes of Health Research Sensorimotor Rehabilitation Team Grant and grants from the Institut de réadaptation en déficience physique de Québec, now integrated into the Centre intégré universitaire de santé et de services sociaux de la Capitale-Nationale, and the Centre interdisciplinaire de recherche en réadaptation et intégration sociale.

Background: Amputation of a lower-limb results in a severe decrease of functional mobility that deeply alters independent living. Motor imagery (MI) refers to the mental representation of an action without engaging its actual execution. The repetitive use of MI has been shown to contribute to promote motor recovery and phantom-limb pain alleviation., Aim: Given the importance of invoking accurate images to benefit from MI practice, and considering the link between motor capacities and MI, the present study investigated the effect of a rehabilitation program on MI ability in patients with lower-limb amputation., Design: Observational and longitudinal study., Setting: Patients recruited from the Amputation program at the Institut de Réadaptation en Déficience Physique de Québec (IRDPQ), in Quebec City, Canada., Population: Patients with trans-tibial or transfemoral amputation, following a rehabilitation program as outpatients., Methods: MI ability of the patients was measured at three different time points along the course of physical therapy., Results: The data revealed a positive effect of the rehabilitation program on MI accuracy of locomotor tasks, and greater MI vividness and accuracy for single-joint movements that patients were still able to physically perform., Conclusions: These findings suggest that MI abilities and actual motor performance are mirrored in a congruent fashion., Clinical Rehabilitation Impact: Therapists should consider this critical aspect when including MI practice in rehabilitation programs among patients with lower-limb amputation.

Advances in maternal and perinatal care in developed countries have led to improved health outcomes for children. These changes may have impacted the profile of children with a cerebral palsy (CP) and groups at risk for CP over time. Using data from the Canadian CP Registry, the objectives of this retrospective cohort study were to describe the profile of children with CP in Quebec born between 1999 and 2010 and identify possible temporal variation in CP risk factors and phenotypic profile. Our sample consisted of 662 children with CP in Quebec. No change in profile or associated risk factors was observed across the birth cohorts 1999 to 2010. Prematurity remains the largest risk factor for CP in Quebec, and children with CP have multiple comorbidities that contribute to overall CP burden. CP registries offer a unique platform to study spectrum disorders and their longitudinal changes over time.

An ethanolic extract from Rhodomyrtus tomentosa leaves (RTL) was studied as a natural alternative to control Staphylococcus aureus, which is an important pathogen responsible for bovine mastitis. The minimal inhibitory concentrations (MICs) of the RTL extract and of rhodomyrtone, a pure compound isolated from the plant, were determined by a microdilution method. Rhodomyrtone and the RTL extract exhibited antibacterial activity against S. aureus, including its persistent phenotype (SCV: small-colony variant) and a biofilm hyperproducer strain, with MICs of 0.25-0.5 and 8-16 µg/mL, respectively. Time-kill kinetics showed a strong bactericidal activity for both the RTL extract- and rhodomyrtone-treated bacteria at 2 × MIC as early as 4 h post-exposure. An additive effect of the extract at 0.5 × MIC was observed in a combination with oxytetracycline or pirlimycin against S. aureus by showing a 64- to 128-fold reduction in antibiotic MICs. Moreover, the RTL extract significantly decreased the number of intracellular SCVs inside bovine mammary epithelial cells. However, the extract or its combination with pirlimycin only slightly improved the activity of pirlimycin against the bacterial colonization of mouse mammary glands. In vitro MICs determined in the presence of casein indicated that the limited activity of the RTL extract in the murine model of mastitis could be linked to neutralization of active components by milk proteins. While the RTL extract showed interesting antibacterial properties in vitro, to be considered as an alternative to antibiotics in dairy farms, formulation studies are needed to cope with the observed reduction of activity in vivo.

Bovine mastitis affects animal health and welfare and milk production and quality, and it challenges the economic success of dairy farms. Staphylococcus aureus is one of the most commonly found pathogens in clinical mastitis but it also causes subclinical, persistent, and difficult-to-treat intramammary infections. Because of the failure of conventional antibiotic treatments and increasing pressure and concern from experts and consumers over the use of antibiotics in the dairy industry, many attempts have been made over the years to develop a vaccine for the prevention and control of Staph. aureus intramammary infections. Still, no commercially available vaccine formulation demonstrates sufficient protection and cost-effective potential. Multiple factors account for the lack of protection, including inadequate vaccine targets, high diversity among mastitis-provoking strains, cow-to-cow variation in immune response, and a failure to elicit an immune response that is appropriate for protection against a highly complex pathogen. The purpose of this review is to summarize key concepts related to the pathogenesis of Staph. aureus, and its interaction with the host, as well as to describe recent vaccine development strategies for prevention and control of Staph. aureus mastitis., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

To reduce the use of antibiotics in animal agriculture, a number of effective or commercially viable alternatives have been implemented by food animal producers or are under development. Perhaps the most well-established strategies are flock and herd management practices to mitigate disease introduction and spread, and, subsequently, reduce the need for antibiotic use. While vaccines in food animal production have been used to prevent both bacterial and viral diseases, but historically, most vaccines have targeted viral diseases. Though vaccines against viral diseases can help reduce the need for antibiotic use by controlling the spread of secondary bacterial infections, more recent vaccines under development specifically target bacteria. New developments in selecting and potentially tailoring bacteriophages provide a promising avenue for controlling pathogenic bacteria without the need for traditional small-molecule antibiotics. In this article we discuss these established and emerging strategies, which are anticipated to reduce the reliance on antibiotics in food animal production and should reduce the prevalence and transmission to humans of antimicrobial resistant bacteria from these systems., (© 2019 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.)

Tebipenem (SPR859) is the microbiologically active form of SPR994 (tebipenem-pivoxil), an orally available carbapenem with activity against extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae Measurement of the relative binding of SPR859 to the bacterial cell targets revealed that it is a potent inhibitor of multiple penicillin-binding proteins (PBPs) but primarily a Gram-negative PBP 2 inhibitor, similar to other compounds in this class. These data support further clinical development of SPR994., (Copyright © 2019 American Society for Microbiology.)

Purpose: The purpose of this proof-of-principle study was to show that virtual reality (VR) technology could be coupled with a self-paced treadmill to further improve walking competency in individuals with chronic stroke. Method: A 62-year-old man with a chronic right hemispheric stroke participated in a treadmill walking programme involving first a control (CTL) protocol, then VR training. In CTL training, he walked without time constraints while viewing still pictures and reacting to treadmill movements similar to those that he would have experienced later in VR training. In VR training, he experienced treadmill movements programmed to simulate changes encountered in five virtual environments rear-projected onto a large screen. Training difficulty in nine sessions over 3 weeks was increased by varying the time constraints, terrain surface changes, and obstacles to avoid. Effects on walking competency were assessed using clinical measures (5 m walk test, 6 min walk test, Berg Balance Scale, Activities-specific Balance Confidence scale) and questionnaires (Assessment of Life Habits Scale and personal appraisal). Results: CTL and VR training resulted in a similar progression through the training sessions of total time walked on the treadmill. The VR training led to an additional increase in speed as measured by walking 5 metres as fast as possible and distance walked in 6 minutes, as well as improved balance self-efficacy and anticipatory locomotor adjustments. As reported by the participant, these improved outcomes transferred to real-life situations. Conclusions: Despite the limited potential for functional recovery from chronic stroke, an individual can achieve improvements in mobility and self-efficacy after participating in VR-coupled treadmill training, compared with treadmill training with the same intensity and surface perturbations but without VR immersion. A larger scale, randomized controlled trial is warranted to determine the efficacy of VR-coupled treadmill training for mobility intervention post-stroke.

Clinical mastitis (CM) is one of the most frequent and costly diseases in dairy cows. A frustrating aspect of CM is its recurrent nature. This review was conducted to synthesize knowledge on risk of repeated cases of CM, effects of recurrent CM cases, and risk factors for CM recurrence. A systematic review methodology was used to identify articles for this narrative review. Searches were performed to identify relevant scientific literature published after 1989 in English or French from 2 databases (PubMed and CAB Abstracts) and 1 search platform (Web of Science). Fifty-seven manuscripts were selected for qualitative synthesis according to the inclusion criteria. Among the 57 manuscripts selected in this review, a description of CM recurrence, its risk factors, and effects were investigated and reported in 33, 37, and 19 selected manuscripts, respectively. Meta-analysis and meta-regression analyses were used to compute risk ratio comparing risk of CM in cows that already had 1 CM event in the current lactation with risk of CM in healthy cows. For these analyses, 9 manuscripts that reported the total number of lactations followed and the number of lactations with ≤1 and ≤2 CM cases were used. When summarizing results from studies requiring ≥5 d between CM events to consider a CM event as a new case, we observed no significant change in CM susceptibility following a first CM case (risk ratio: 0.99; 95% confidence interval: 0.86-1.14). However, for studies using a more liberal CM recurrence definition (i.e., only 24 h between CM events to consider new CM cases), we observed a 1.54 times greater CM risk (95% confidence interval: 1.20-1.97) for cows that already had 1 CM event in the current lactation compared with healthy cows. The most important risk factors for CM recurrence were parity (i.e., higher risk in older cows), a higher milk production, pathogen species involved in the preceding case, and whether a bacteriological cure was observed following the preceding case. The most important effects of recurrent CM were the milk yield reduction following a recurrent CM case, which was reported to be similar to that of the first CM case, and the increased risk of culling and mortality, which were reported to surpass those of first CM cases., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro -generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10 5 -fold for FC04-100., (Copyright © 2018 American Society for Microbiology.)

Background: Tomatidine (TO) is a plant steroidal alkaloid that possesses an antibacterial activity against the small colony variants (SCVs) of Staphylococcus aureus. We report here the spectrum of activity of TO against other species of the Bacillales and the improved antibacterial activity of a chemically-modified TO derivative (FC04-100) against Listeria monocytogenes and antibiotic multi-resistant S. aureus (MRSA), two notoriously difficult-to-kill microorganisms., Methods: Bacillus and Listeria SCVs were isolated using a gentamicin selection pressure. Minimal inhibitory concentrations (MICs) of TO and FC04-100 were determined by a broth microdilution technique. The bactericidal activity of TO and FC04-100 used alone or in combination with an aminoglycoside against planktonic bacteria was determined in broth or against bacteria embedded in pre-formed biofilms by using the Calgary Biofilm Device. Killing of intracellular SCVs was determined in a model with polarized pulmonary cells., Results: TO showed a bactericidal activity against SCVs of Staphylococcus aureus, Bacillus cereus, B. subtilis and Listeria monocytogenes with MICs of 0.03-0.12 μg/mL. The combination of an aminoglycoside and TO generated an antibacterial synergy against their normal phenotype. In contrast to TO, which has no relevant activity by itself against Bacillales of the normal phenotype (MIC > 64 μg/mL), the TO analog FC04-100 showed a MIC of 8-32 μg/mL. Furthermore, FC04-100 showed a strong bactericidal activity against L. monocytogenes SCVs in kill kinetics experiments, while TO did not. The addition of FC04-100 (4 μg/mL) to a cefalexin:kanamycin (3:2) combination improved the activity of the combination by 32 fold against cefalexin and kanamycin-resistant MRSA strains. In combination with gentamicin, FC04-100 also exhibited a strong bactericidal activity against biofilm-embedded S. aureus. Also, FC04-100 and TO showed comparable intracellular killing of S. aureus SCVs., Conclusions: Chemical modifications of TO allowed improvement of its antibacterial activity against prototypical S. aureus and of its bactericidal activity against L. monocytogenes. Antibacterial activities against such prominent pathogens could be useful to prevent Listeria contamination in the food chain or as treatment for MRSA infections.