Search

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised. more...

The entry routes and translocation mechanisms of bacterial pathogens into the central nervous system remain obscure. We report here thatStreptococcus pneumoniae(Sp) or polystyrene microspheres, applied to the nose of a mouse, appeared in the meninges of the dorsal cortex within minutes. Recovery of viable bacteria from dissected tissue and fluorescence microscopy showed that up to at least 72h, Sp and microspheres were predominantly in the outer of the two meninges, the pachymeninx. No Sp were found in blood or cerebrospinal fluid. Evidence that this was not an artifact of the method of administration is that in mice infected by horizontal transmission, Sp were also predominantly in the meninges and absent from blood. Intravital imaging through the skull, and flow cytometry showed recruitment and activation of LysM+cells in the dorsal pachymeninx at 5h and 10h following intranasal infection. Imaging of the cribriform plate suggested that both Sp and microspheres entered through its foramina via an inward flow of fluid connecting the nose to the pachymeninx. Our findings bring further insight into the invasion mechanisms of bacterial pathogens such as Sp into the central nervous system, but are also pertinent to the delivery of drugs to the brain, and the entry of air-borne particles into the cranium. more...

Staphylococcus aureus nasal colonization is a risk factor for infection. A large proportion of the population are identified as potential S. aureus carriers yet we only partially understand the repertoire of genetic factors that promote long-term nasal colonization. Here we present a novel murine model of nasopharyngeal colonization that requires a low S. aureus inoculum and is amenable to experimental evolution approaches. We used this model to experimentally evolve S. aureus using successive passages in the nasopharynx to identify those genetic loci under selection. After 3 cycles of colonization, mutations were identified in mannitol, sorbitol, arginine, nitrite and lactate metabolism genes promoting key pathways in nasal colonization. Stress responses were identified as being under selective pressure, with mutations in DNA repair genes including dnaJ and recF and key stress response genes clpL, rpoB and ahpF. Peptidoglycan synthesis pathway genes also revealed mutations indicating potential selection for alteration of the cell surface. The infection model used here is versatile to assist decolonization and persistence studies. more...

Streptococcus pyogenes (GAS) is among the most diverse of all human pathogens, responsible for a range of clinical manifestations, from mild superficial infections such as pharyngitis to serious invasive infections such as necrotising fasciitis and sepsis. The drivers of these different disease phenotypes are not known. The GAS cholesterol-dependent cytolysin, streptolysin O (SLO), has well established cell and tissue destructive activity. We investigated the role of SLO in determining disease outcomein vivo, by using two different clinical lineages; the recently emerged hypervirulent outbreakemmtype 32.2 strains, which result in sepsis, and theemmtype 1.0 strains which cause septic arthritis. Using clinically relevantin vivomouse models of sepsis and a novel septic arthritis model, we demonstrated that the amount and activity of SLO is vital in determining the pathotype of infection. Theemm32.2 strain produced large quantities of highly haemolytic SLO that resulted in rapid development of sepsis. By contrast, the lower levels and haemolytic activity ofemm1.0 SLO led to translocation of bacteria to joints. Importantly, sepsis associated strains that were attenuated by deletion or inhibition of SLO also translocated to the joint, confirming the key role of SLO in determining infection niche. Our findings demonstrate that SLO is key toin vivopathotype and disease outcome. Careful consideration should be given to novel therapy or vaccination strategies that target SLO. Whilst neutralising SLO activity may reduce severe invasive disease, it has the potential to promote chronic inflammatory conditions such as septic arthritis. more...

Prolonging the clinical effectiveness of {beta}-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 gene re-sensitizes MRSA to {beta}-lactam antibiotics. The cycA mutation also resulted in hyper-susceptibility to DCS, an alanine analogue antibiotic that inhibits the alanine racemase and D-Alanine ligase enzymes required for D-Alanine incorporation into peptidoglycan. Amino acid transport studies showed that CycA functions as an alanine permease, and the cycA mutation was accompanied by increased accumulation of muropeptides with tripeptide stems lacking the terminal D-Ala-D-Ala, and reduced peptidoglycan cross linking. Exposure of MRSA to DCS was also associated with a dose-dependent accumulation of muropeptides with tripeptide stems and reduced PG crosslinking. Because impaired alanine transport or DCS-treatment have similar effects on PG structure, synergism between {beta}-lactams and DCS was investigated. Therapeutically achievable concentrations of DCS re-sensitised MRSA to {beta}-lactam antibiotics in vitro and significantly enhanced MRSA eradication in a mouse bacteraemia model. The susceptibility of several other ESKAPE group pathogens to {beta}-lactams was also increased by DCS. These data reveal the potential of DCS, or new drugs targeting CycA, to facilitate the reintroduction of {beta}-lactam antibiotics for the treatment of infections caused by MRSA.nnAuthor SummaryTreatment options for infections caused by ESKAPE group pathogens continue to dwindle with increasing antimicrobial resistance. Particularly important are the {beta}-lactams, which remain first line antibiotics for many infections, and finding new ways to maintain their clinical effectiveness is a priority. Here we report that mutation of cycA in MRSA, which was found to encode an alanine permease, was associated with hyper-susceptibility to {beta}-lactam antibiotics and D-cycloserine (DCS), used in the treatment of multi-drug resistant tuberculosis infections. DCS re-sensitised MRSA to oxacillin and other {beta}-lactams in vitro and significantly enhanced oxacillin activity in a mouse model of bacteraemia. DCS also sensitised other ESKAPE pathogens to {beta}-lactams revealing the therapeutic potential of DCS as an adjunct drug for these important antibiotics. more...