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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

This study investigated the antibacterial activity of the plant alkaloid tomatidine (TO) against Staphylococcus aureus grown in the presence of Pseudomonas aeruginosa . Since the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline- N -oxide (HQNO) is known to cause a respiratory deficiency in S. aureus and respiratory-deficient S. aureus are known to be hypersensitive to TO, we assessed kill kinetics of TO (8 μg/ml) against S. aureus in coculture with P. aeruginosa . Kill kinetics were also assessed using P. aeruginosa mutants deficient in the production of different exoproducts and quorum sensing-related compounds. After 24 h in coculture, TO increased the killing of S. aureus by 3.4 log 10 CFU/ml in comparison to that observed in a coculture without TO. The effect of TO was abolished when S. aureus was in coculture with the lasR rhlR , pqsA , pqsL , or lasA mutant of P. aeruginosa . The bactericidal effect of TO against S. aureus in coculture with the pqsL mutant was restored by supplemental HQNO. In an S. aureus monoculture, the combination of HQNO and TO was bacteriostatic, indicating that the pqsL mutant produced an additional factor required for the bactericidal effect. The bactericidal activity of TO was also observed against a tobramycin-resistant methicillin-resistant S. aureus (MRSA) in coculture with P. aeruginosa , and the addition of tobramycin significantly suppressed the growth of both microorganisms. TO shows a strong bactericidal effect against S. aureus when cocultured with P. aeruginosa . The combination of TO and tobramycin may represent a new treatment approach for cystic fibrosis patients frequently cocolonized by MRSA and P. aeruginosa .

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.

Objectives This study characterized the multiple biological activities of the natural compound tomatidine against Staphylococcus aureus. Notably, this work examined the antibacterial activity of tomatidine in combination with other antibiotics and the influence of this compound on the expression of virulence factors in S. aureus. Methods The effect of tomatidine on the susceptibility of S. aureus to several antibiotic classes was determined by a broth microdilution procedure and a chequerboard protocol to measure fractional inhibitory concentration indices and to reveal drug interactions. Time-kill experiments for aminoglycoside/tomatidine combinations were also performed. The haemolytic ability of several strains in the presence of tomatidine was measured on blood agar plates and the expression of virulence-associated genes in strain ATCC 29213 treated with tomatidine was monitored by quantitative PCR. Results Tomatidine specifically potentiated the inhibitory effect of aminoglycosides but not of other classes of drugs. This potentiating effect was observed against strains of different clinical origins (human blood, cystic fibrosis airways, osteomyelitis, skin tissues and bovine mastitis), including aminoglycoside-resistant bacteria possessing the aac(6')-aph(2″), ant(4')-Ia and aph(3')-IIIa genes. The killing kinetics for the combination of aminoglycosides with tomatidine revealed strong bactericidal activity. Although tomatidine did not possess growth-inhibitory activity of its own against prototypical S. aureus, it inhibited the haemolytic activity of several strains and, more specifically, blocked the expression of several genes normally influenced by the agr system. Conclusions These results show that tomatidine is an aminoglycoside potentiator that also acts as an anti-virulence agent targeting both antibiotic-susceptible and antibiotic-resistant S. aureus.

Primary cesium frequency standards realized as cesium fountains offer the prospect of frequency accuracy in the 10-15 range, or better. Active hydrogen masers are good candidates for the source of a local time scale to disseminate frequency at this level of accuracy after calibration by a cesium fountain that operates intermittently. We develop a procedure for evaluating the frequency uncertainty associated with the resulting calibrated time scale. We apply the procedure to the expected performance of masers and cesium fountains, and find that current hydrogen masers can be used to deliver frequency uncertainties of less than 10-15, and might be used to approach 10-16. The procedure is appropriate to use, for example, when reporting uncertainties for cesium fountain average frequencies delivered to assist the International Bureau of Weights and Measures (BIPM) in steering International Atomic Time (TAI).

Three new primary cesium clocks with an interaction length of 1 m are now in operation at the National Research Council (NRC). Used as secondary clocks since late 1978 and as primary clocks since December 1979, they have an expected lifetime of up to 25 years, an accuracy limit of 1.5 X 10-13, a 24-h frequency stability ?(2, ?) of less than 2 X 10-14, and agree in frequency to better than I X 10-13 with the NRC primary clock CsV.

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.

Background Cranberry fruits possess many biological activities partly due to their various phenolic compounds; however the underlying modes of action are poorly understood. We studied the effect of cranberry fruit extracts on the gene expression of Staphylococcus aureus to identify specific cellular processes involved in the antibacterial action. Methods Transcriptional profiles of four S. aureus strains grown in broth supplemented or not with 2 mg/ml of a commercial cranberry preparation (Nutricran®90) were compared using DNA arrays to reveal gene modulations serving as markers for biological activity. Ethanol extracted pressed cakes from fresh fruits also produced various fractions and their effects on marker genes were demonstrated by qPCR. Minimal inhibitory concentrations (MICs) of the most effective cranberry fraction (FC111) were determined against multiple S. aureus strains and drug interactions with β-lactam antibiotics were also evaluated. Incorporation assays with [3H]-radiolabeled precursors were performed to evaluate the effect of FC111 on DNA, RNA, peptidoglycan (PG) and protein biosynthesis. Results Treatment of S. aureus with Nutricran®90 or FC111 revealed a transcriptional signature typical of PG-acting antibiotics (up-regulation of genes vraR/S, murZ, lytM, pbp2, sgtB, fmt). The effect of FC111 on PG was confirmed by the marked inhibition of incorporation of D-[3H]alanine. The combination of β-lactams and FC111 in checkerboard assays revealed a synergistic activity against S. aureus including strain MRSA COL, which showed a 512-fold drop of amoxicillin MIC in the presence of FC111 at MIC/8. Finally, a therapeutic proof of concept was established in a mouse mastitis model of infection. S. aureus-infected mammary glands were treated with amoxicillin, FC111 or a combination of both; only the combination significantly reduced bacterial counts from infected glands (P Conclusions The cranberry fraction FC111 affects PG synthesis of S. aureus and acts in synergy with β-lactam antibiotics. Such a fraction easily obtained from poorly exploited press-cake residues, may find interesting applications in the agri-food sector and help reduce antibiotic usage in animal food production.