Fructose transport-deficient Staphylococcus aureus reveals important role of epithelial glucose transporters in limiting sugar-driven bacterial growth in airway surface liquid.

Hyperglycaemia as a result of diabetes mellitus or acute illness is associated with increased susceptibility to respiratory infection with Staphylococcus aureus. Hyperglycaemia increases the concentration of glucose in airway surface liquid (ASL) and promotes the growth of S. aureus in vitro and in vivo. Whether elevation of other sugars in the blood, such as fructose, also results in increased concentrations in ASL is unknown and whether sugars in ASL are directly utilised by S. aureus for growth has not been investigated. We obtained mutant S. aureus JE2 strains with transposon disrupted sugar transport genes. NE768(fruA) exhibited restricted growth in 10 mM fructose. In H441 airway epithelial-bacterial co-culture, elevation of basolateral sugar concentration (5-20 mM) increased the apical growth of JE2. However, sugar-induced growth of NE768(fruA) was significantly less when basolateral fructose rather than glucose was elevated. This is the first experimental evidence to show that S. aureus directly utilises sugars present in the ASL for growth. Interestingly, JE2 growth was promoted less by glucose than fructose. Net transepithelial flux of D-glucose was lower than D-fructose. However, uptake of D-glucose was higher than D-fructose across both apical and basolateral membranes consistent with the presence of GLUT1/10 in the airway epithelium. Therefore, we propose that the preferential uptake of glucose (compared to fructose) limits its accumulation in ASL. Pre-treatment with metformin increased transepithelial resistance and reduced the sugar-dependent growth of S. aureus. Thus, epithelial paracellular permeability and glucose transport mechanisms are vital to maintain low glucose concentration in ASL and limit bacterial nutrient sources as a defence against infection

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

Staphylococcus aureus infection dynamics

Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed

Staphylococcus aureus forms spreading dendrites that have characteristics of active motility

Staphylococcus aureus is historically regarded as a non-motile organism. More recently it has been shown that S. aureus can passively move across agar surfaces in a process called spreading. We re-analysed spreading motility using a modified assay and fo- cused on observing the formation of dendrites: branching structures that emerge from the central colony. We discovered that S. aureus can spread across the surface of media in struc- tures that we term ‘comets’, which advance outwards and precede the formation of dendrites. We observed comets in a diverse selection of S. aureus isolates and they exhibit the following behaviours: (1) They consist of phenotypically distinct cores of cells that move forward and seed other S. aureus cells behind them forming a comet ‘tail’; (2) they move when other cells in the comet tail have stopped moving; (3) the comet core is held together by a matrix of slime; and (4) the comets etch trails in the agar as they move forwards. Comets are not con- sistent with spreading motility or other forms of passive motility. Comet behaviour does share many similarities with a form of active motility known as gliding. Our observations therefore suggest that S. aureus is actively motile under certain conditions

Evolution of metabolic divergence in <i>Pseudomonas aeruginosa</i> during long-term infection facilitates a proto-cooperative interspecies interaction

The effect of polymicrobial interactions on pathogen physiology and how it can act either to limit pathogen colonization or to potentiate pathogen expansion and virulence are not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found together in polymicrobial human infections. However, we have previously shown that the interactions between these two bacterial species are strain dependent. Whereas P. aeruginosa PAO1, a commonly used laboratory strain, effectively suppressed S. aureus growth, we observed a commensal-like interaction between the human host-adapted strain, DK2-P2M24-2003, and S. aureus. In this study, characterization by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) and mass spectral (MS) molecular networking revealed a significant metabolic divergence between P. aeruginosa PAO1 and DK2-P2M24-2003, which comprised several virulence factors and signaling 4-hydroxy-2-alkylquinoline (HAQ) molecules. Strikingly, a further modulation of the HAQ profile was observed in DK2-P2M24-2003 during interaction with S. aureus, resulting in an area with thickened colony morphology at the P. aeruginosa–S. aureus interface. In addition, we found an HAQ-mediated protection of S. aureus by DK2-P2M24-2003 from the killing effect of tobramycin. Our findings suggest a model where the metabolic divergence manifested in human host-adapted P. aeruginosa is further modulated during interaction with S. aureus and facilitate a proto-cooperative P. aeruginosa–S. aureus relationship

Defining motility in the Staphylococci

The ability of bacteria to move is critical for their survival in diverse environments and multiple ways have evolved to achieve this. Two forms of motility have recently been described for Staphylococcus aureus, an organism previously considered to be non-motile. One form is called spreading, which is a type of sliding motility and the second form involves comet formation, which has many observable characteristics associated with gliding motility. Darting motility has also been observed in Staphylococcus epidermidis. This review describes how motility is defined and how we distinguish between passive and active motility. We discuss the characteristics of the various forms of Staphylococci motility, the molecular mechanisms involved and the potential future research directions

MASTL overexpression promotes chromosome instability and metastasis in breast cancer.

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival

Infective endocarditis caused by Salmonella enteritidis in a dialysis patient: a case report and literature review

BackgroundInfective endocarditis is significantly more common in haemodialysis patients as compared with the general population, the causative pathogen is generally Staphylococcus aureus; there have been no previously reported cases of infective endocarditis caused by a Salmonella species in haemodialysis patients.Case presentationWe report the case of a 68 year-old woman on haemodialysis who developed infective endocarditis as a result of Salmonella enteritidis. Although we treated the patient with ceftriaxone combined with ciprofloxacin, infective endocarditis was not detected early enough and unfortunately developed into cerebral septic emboli, which ultimately resulted in death.ConclusionAlthough there are several reports that Salmonella endocarditis without cardiac failure can be successfully treated with antibiotics alone, early surgical intervention is essential for some cases to prevent life-threatening complications. Transesophageal echocardiography should be performed in any patient with high clinical suspicion of infective endocarditis. To the best of our knowledge, this is the first case-report of Salmonella endocarditis in a haemodialysis patient

Impaired alanine transport or exposure to d-cycloserine increases the susceptibility of MRSA to β-lactam antibiotics

Prolonging the clinical effectiveness of β-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA re-sensitized MRSA to β-lactam antibiotics. The cycA mutation also resulted in hyper-susceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan (PG). Alanine transport was impaired in the cycA mutant and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced PG crosslinking, prompting us to investigate synergism between β-lactams and DCS. DCS re-sensitised MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteraemia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics.</jats:p

Epidemiology of community-onset Staphylococcus aureus infections in pediatric patients: an experience at a Children's Hospital in central Illinois

<p>Abstract</p> <p>Background</p> <p>The nation-wide concern over methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) has prompted many clinicians to use vancomycin when approaching patients with suspected staphylococcal infections. We sought to characterize the epidemiology of community-onset <it>S. aureus </it>infections in hospitalized children to assist local clinicians in providing appropriate empiric antimicrobial therapy.</p> <p>Methods</p> <p>From January 2005–June 2008, children (0–18 years old) admitted to the Children's Hospital of Illinois with community-onset <it>S. aureus </it>infections were identified by a computer-assisted laboratory-based surveillance and medical record review.</p> <p>Results</p> <p>Of 199 patients, 67 (34%) had invasive infections, and 132 (66%) had skin and soft tissue infections (SSTIs). Among patients with invasive infections, <it>S. aureus </it>isolates were more likely to be susceptible to methicillin (MSSA 63% vs. MRSA 37%), whereas patients with SSTIs, <it>S. aureus </it>isolates were more likely to be resistant to methicillin (MRSA 64% vs. MSSA 36%). Bacteremia and musculoskeletal infections were the most common invasive infections in both groups of <it>S. aureus</it>. Pneumonia with empyema was more likely to be caused by MRSA (<it>P </it>= 0.02). The majority (~90%) of MRSA isolates were non-multidrug resistant, even in the presence of healthcare-associated risk factors.</p> <p>Conclusion</p> <p>Epidemiological data at the local level is important for antimicrobial decision-making. MSSA remains an important pathogen causing invasive community-onset <it>S. aureus </it>infections among hospitalized children. In our hospital, nafcillin in combination with vancomycin is recommended empiric therapy in critically ill patients with suspected invasive staphylococcal infections. Because up to 25% of MSSA circulating in our area are clindamycin-resistant, clindamycin should be used cautiously as empiric monotherapy in patients with suspected invasive staphylococcal infections.</p