Mode of Antibacterial Action of Tomatidine C3-Diastereoisomers.

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 3ß-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, TM-02 (C3-ß) 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 Ki 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-ß-diastereoisomers may have more specific antibacterial action than C3-α.

From garden to lab: C-3 chemical modifications of tomatidine unveil broad-spectrum ATP synthase inhibitors to combat bacterial resistance.

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.

Rationally Designed Pyrimidine Compounds: Promising Novel Antibiotics for the Treatment of Staphylococcus aureus-Associated Bovine Mastitis.

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.

The Therapeutic Value of Solanum Steroidal (Glyco)Alkaloids: A 10-Year Comprehensive Review.

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.

Chronic Diarrhea Caused by a Klebsiella oxytoca Toxin Producer Strain Following Antibiotic-Associated Hemorrhagic Colitis: Successful Treatment by Fecal Microbiota Transplant.

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.

Staphylococcus aureus Small-Colony Variants from Airways of Adult Cystic Fibrosis Patients as Precursors of Adaptive Antibiotic-Resistant Mutations.

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 (ΔhemB) 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.

Effective Treatment of Staphylococcus aureus Intramammary Infection in a Murine Model Using the Bacteriophage Cocktail StaphLyse™.

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.

One Health-One Biofilm.

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.

Comparative genomic analysis of Staphylococcus aureus isolates associated with either bovine intramammary infections or human infections demonstrates the importance of restriction-modification systems in host adaptation.

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.

Secondary Staphylococcus aureus intramammary colonization is reduced by non-aureus staphylococci exoproducts.

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.

Susceptibility to Nisin, Bactofencin, Pediocin and Reuterin of Multidrug Resistant Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus uberis Causing Bovine Mastitis.

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.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models.

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 ß-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-1ß 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.

Immune and experimental infection responses of dairy cows vaccinated with the combination of six Staphylococcus aureus proteins that are expressed during bovine intramammary infection and a triple adjuvant.

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.

Counteracting Bacterial Motility: A Promising Strategy to Narrow Listeria monocytogenes Biofilm in Food Processing Industry.

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 (ZnCl2), 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.

In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics.

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.

A Siderophore Analog of Fimsbactin from Acinetobacter Hinders Growth of the Phytopathogen Pseudomonas syringae and Induces Systemic Priming of Immunity in Arabidopsis thaliana.

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 syringaepv 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.

Bactericidal Activity of the Bacterial ATP Synthase Inhibitor Tomatidine and the Combination of Tomatidine and Aminoglycoside Against Persistent and Virulent Forms of Staphylococcus aureus.

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.

Prevalence and characteristics of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) isolated from chicken meat in the province of Quebec, Canada.

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.

Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus.

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.

Evaluation of a Rhodomyrtus tomentosa ethanolic extract for its therapeutic potential on Staphylococcus aureus infections using in vitro and in vivo models of mastitis.

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.