The widespread use of antibiotics has significantly increased the number of resistant bacteria, which has also increased the urgency of rapid bacterial detection and profiling their antibiotic response. Current clinical methods for antibiotic susceptibility testing (AST) rely on culture and require at least 16 to 24 h to conduct. Therefore, there is an urgent need for a rapid method that can test the susceptibility of bacteria in a culture-free manner. Here we demonstrate a rapid AST method by monitoring the glucose metabolic activity of live bacteria at the single-cell level with hyperspectral stimulated Raman scattering (SRS) imaging. Using vancomycin-susceptible and -resistant enterococci E. faecalis as models, we demonstrate that the metabolic uptake of deuterated glucose in a single living bacterium can be quantitatively monitored via hyperspectral SRS imaging. Remarkably, the metabolic activity of susceptible bacteria responds differently to antibiotics from the resistant strain within only 0.5 h from the addition of antibiotics. Therefore, bacterial susceptibility and the minimum inhibitory concentration (MIC) of antibiotics can be determined within one cell cycle. Our metabolic imaging method is applicable to other bacteria species including E. coli, K. Pneumoniae, and S. aureus as well as different antibiotics, regardless of their mechanisms of inhibiting or killing bacteria.

The objective of this work was to evaluate extrusion cooking as a means to improve the nutritional properties of Phaseolus vulgaris L. that had been stored either at 42 degrees C and 80% relative humidity for 6 weeks or for periods >1 year in cereal stores in tropical conditions. Storage under these conditions resulted in an increase in cooking time increased (7.7- and 12-fold, respectively) as a result of development of the hard-to-cook (HTC) defect. Single-screw extrusion of the milled beans was carried out at four barrel temperatures and two moisture contents. The extrudate bulk density and water solubility index decreased with increasing temperature, whereas the water absorption index increased due to the higher proportion of gelatinized starch in the extruded samples. Both fresh and HTC beans contained nutritionally significant amounts of lectins, trypsin, and alpha-amylase inhibitors, which were mostly inactivated by extrusion. Extrusion also caused a considerable redistribution of insoluble dietary fiber to soluble, although the total dietary fiber content was not affected. Changes in solubility involved pectic polysaccharides, arabinose and uronic acids being the main sugars involved. Stored beans subjected to extrusion cooking showed physical and chemical characteristics similar to those of extrudates from fresh beans.

No abstract

Candida albicans is the single most prevalent cause of fungal bloodstream infections worldwide causing significant mortality as high as 50 percent. This high mortality rate is, in part, due to the inability to initiate an effective antifungal therapy early in the disease process. Mortality rates significantly increase after 12 hours of delay in initiating the appropriate antifungal therapy following a positive blood culture. Early administration of appropriate antifungal therapy is hampered by the slow turnovers of the conventional antimicrobial testing techniques, which require days of incubation. To address this unmet need, we explored the potential of employing stimulated Raman scattering (SRS) imaging to probe for metabolic differences between fluconazole-susceptible and -resistant strains at a single cell level in search of a metabolic signature. Metabolism is integral to pathogenicity. Since only a few hours are needed to observe a full metabolic cycle in C. albicans, metabolic profiling provides an avenue for rapid antimicrobial susceptibility testing. C-H frequency (2850 cm) SRS imaging revealed a substantial difference in lipogenesis between the fluconazole-susceptible and -resistant C. albicans. Exposure to fluconazole, an antimicrobial drug that targets ergosterol biosynthesis, only affected the lipogenesis in the susceptible strain. These results show that single cell metabolic imaging via SRS microscopy can be used for rapid detection of antimicrobial susceptibility.

The rise of antibiotic resistance, especially in Staphylococcus aureus,and the increasing deathrate due to multiresistant bacteria have been well documented. The need for new chemical entitiesa nd/or the identification of novel targets for antibacterial drug development is high. Lipoteichoic acid (LTA), am embrane-attached anionic polymer,i si mportant for the growth and virulence of many Gram-positive bacteria, and interest has been high in the discovery of LTAb iosynthesis inhibitors. Thus far,o nly ah andfulo fL TA biosynthesis inhibitors have been described with moderate( MIC = 5.34 mgmL À1 )t ol ow (MIC = 1024 mgmL À1 )a ctivities against S. aureus. Herein we describe the identification of novel compounds that potently inhibit LTA biosynthesis in S. aureus,d isplaying impressive antibacterial activities (MIC as low as 0.25 mgmL À1 )a gainst methicillin-resistant S. aureus (MRSA). Under similari nvitro assay conditions, these compounds are 4-fold more potent than vancomycin and 8fold more potent than linezolid against MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are still difficult to treat, despite the availability of many FDA-approved antibiotics. Thus, new compound scaffolds are still needed to treat MRSA. The oxadiazole-containing compound, HSGN-94, has been shown to reduce lipoteichoic acid (LTA) in S. aureus, but the mechanism that accounts for LTA biosynthesis inhibition remains uncharacterized. Herein, we report the elucidation of the mechanism by which HSGN-94 inhibits LTA biosynthesis via utilization of global proteomics, activity-based protein profiling, and lipid analysis via multiple reaction monitoring (MRM). Our data suggest that HSGN-94 inhibits LTA biosynthesis via direct binding to PgcA and downregulation of PgsA. We further show that HSGN-94 reduces the MRSA load in skin infection (mouse) and decreases pro-inflammatory cytokines in MRSA-infected wounds. Collectively, HSGN-94 merits further consideration as a potential drug for staphylococcal infections.

No abstract

Immune cells sense bacteria-derived c-di-GMP and c-di-AMP as well as host-derived cGAMP, which is synthesized by cGAS upon binding to the pathogen’s DNA, to mount an immunological response (cytokine production) via the STING-TBK1 pathway. Successful pathogens, such as Mycobacterium tuberculosis and group B streptococcus, harbor phos­pho­di­esterases (PDEs) that can cleave bacterial c-di-AMP as well as host-derived cGAMP to blunt the host’s response to infection. Selective inhibitors of bacterial cyclic dinucleotide (CDN) PDEs are needed as tool compounds to study the role(s) of CDN PDEs during infection and they could also become bona fide antivirulence compounds, but there is a paucity of such compounds. Using a high-throughput assay, we identified six inhibitors of MTB CDN PDE (CdnP). The most potent inhibitor, C82 with an IC50 of ∼18 μM, did not inhibit the enzymatic activities of three other bacterial CDN PDEs (Yybt, RocR, and GBS-CdnP), a viral CDN PDE (poxin) or mammalian ENPP1.