Histidine transport is essential for the growth of Staphylococcus aureus at low pH.

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production.

A Staphylococcus aureus strain deleted for the c-di-AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine-betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild-type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c-di-AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c-di-AMP signalling in S. aureus.

Smelling Pseudomonas aeruginosa infections using a whole-cell biosensor - An alternative for the gold-standard culturing assay.

Improved easy-to-use diagnostic tools for infections are in strong demand worldwide. Yet, despite dramatic advances in diagnostic technologies, the gold-standard remains culturing. Here we offer an alternative tool demonstrating that a bacterial biosensor can efficiently detect Pseudomonas aeruginosa infections in patients suffering from otitis externa. Detection was based on specific binding between the biosensor and 2-aminoacetophenone (2-AA), a volatile produced by P. aeruginosa in high amounts. We collected pus samples from ears of 26 subjects exhibiting symptoms of otitis externa. Detection of P. aeruginosa using the biosensor was compared to detection using gold-standard culturing assay and to gas-chromatograph-mass-spectrometry (GC-MS) analyses of 2-AA. The biosensor strain test matched the culture assay in 24 samples (92%) and the GC-MS analyses in 25 samples (96%). With this result in hand, we designed a device containing a whole-cell luminescent biosensor combined with a photo-multiplier tube. This device allowed detection of 2-AA at levels as low as 2 nmol, on par with detection level of GC-MS. The results of the described study demonstrate that the volatile 2-AA serves as an effective biomarker for P. aeruginosa in ear infections, and that activation of the biosensor strain by 2-AA provides a unique opportunity to design an easy-to-use device that can specifically detect P. aeruginosa infections.

Automatic microfiber filtration (AMF) of surface water: impact on water quality and biofouling evolution.

In the current study we examined the impact of thread filtration using an automatic microfiber filter on Lake Kinneret water quality and as a new application to control biofouling over time. We found that automatic microfiber filtration (AMF) reduced total iron and aluminum in water by over 80%. Particle analysis (>2 µm) revealed a total particle removal efficiency of ≈ 90%, with AMF removal efficiency increasing with increasing particle size and decreasing particle circularity. Regarding microbiological parameters, AMF did not affect bacterial counts or composition in the water. However, it did control biofilm evolution and affected its microbial community composition. AMF controlled biofilm over time by maintaining premature biofilms of less than 10 µm mean thickness compared to biofilms of unfiltered water (up to 60 µm mean thickness). In addition, biofilms developing in AMF filtered water contained relatively low levels of extracellular polymeric substances. While biofilms of unfiltered water were dominated by Proteobacteria (≤ 50%) followed by Bacteroidetes (20-30%) during all 4 weeks of the experiment, biofilms of AMF filtered water were dominated by Proteobacteria (≤ 90%) and especially Alphaproteobacteria after 2 weeks, and Chloroflexi (≈ 60%) after 4 weeks. The decrease in Bacteroidetes might originate from removal of transparent exopolymer particles, which are occasionally colonized by Bacteroidetes. The increase in Alphaproteobacteria and Chloroflexi was explained by these robust groups' ability to adjust to different environments.