Selection of Staphylococcus aureus in a murine nasopharyngeal colonization model.

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 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 murine model used here is versatile to question colonization, persistence and evolution studies. We studied the human pathogen Staphylococcus aureus in our search to determine factors that contribute to its ability to live in the human nose and throat. The anterior nares and nasopharynx are considered primary habitats but we do not understand how the pathogen adapts as it moves from one person to the next. We first determined sustained survival of the pathogen over multiple days in the nasopharynx that might act as a good model for human persistence due to the low numbers of bacteria needed for it to establish. By using successive rounds of colonization of the nasopharynx across different mice we revealed that multiple genetic changes in the S. aureus occurred. These changes were found in genes associated with the cell surface and metabolism and might indicate adaptation to the niche. One gene showed an accumulation of multiple mutations supporting a key contribution in adaptation but the role of the protein it encodes is not yet known. The contribution of these genes and genetic changes are unclear but indicate an area for future research to better understand how this common human pathogen is so successful at human colonization and survival.

Laser-Induced Periodic Surface Structure Enhances Neuroelectrode Charge Transfer Capabilities and Modulates Astrocyte Function.

The brain machine interface (BMI) describes a group of technologies capable of communicating with excitable nervous tissue within the central nervous system (CNS). BMIs have seen major advances in recent years, but these advances have been impeded because of a temporal deterioration in the signal to noise ratio of recording electrodes following insertion into the CNS. This deterioration has been attributed to an intrinsic host tissue response, namely, reactive gliosis, which involves a complex series of immune mediators, resulting in implant encapsulation via the synthesis of pro-inflammatory signaling molecules and the recruitment of glial cells. There is a clinical need to reduce tissue encapsulation in situ and improve long-term neuroelectrode functionality. Physical modification of the electrode surface at the nanoscale could satisfy these requirements by integrating electrochemical and topographical signals to modulate neural cell behavior. In this study, commercially available platinum iridium (Pt/Ir) microelectrode probes were nanotopographically functionalized using femto/picosecond laser processing to generate laser-induced periodic surface structures (LIPSS). Three different topographies and their physical properties were assessed by scanning electron microscopy and atomic force microscopy. The electrochemical properties of these interfaces were investigated using electrochemical impedance spectroscopy and cyclic voltammetry. The in vitro response of mixed cortical cultures (embryonic rat E14/E17) was subsequently assessed by confocal microscopy, ELISA, and multiplex protein array analysis. Overall LIPSS features improved the electrochemical properties of the electrodes, promoted cell alignment, and modulated the expression of multiple ion channels involved in key neuronal functions.

Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to ß-lactam Antibiotics.

Prolonging the clinical effectiveness of ß-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 resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. 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 peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.

Evolutionary trade-offs associated with loss of PmrB function in host-adapted Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonises the upper airway of cystic fibrosis (CF) patients, providing a reservoir of host-adapted genotypes that subsequently establish chronic lung infection. We previously experimentally-evolved P. aeruginosa in a murine model of respiratory tract infection and observed early-acquired mutations in pmrB, encoding the sensor kinase of a two-component system that promoted establishment and persistence of infection. Here, using proteomics, we show downregulation of proteins involved in LPS biosynthesis, antimicrobial resistance and phenazine production in pmrB mutants, and upregulation of proteins involved in adherence, lysozyme resistance and inhibition of the chloride ion channel CFTR, relative to wild-type strain LESB65. Accordingly, pmrB mutants are susceptible to antibiotic treatment but show enhanced adherence to airway epithelial cells, resistance to lysozyme treatment, and downregulate host CFTR expression. We propose that P. aeruginosa pmrB mutations in CF patients are subject to an evolutionary trade-off, leading to enhanced colonisation potential, CFTR inhibition, and resistance to host defences, but also to increased susceptibility to antibiotics.

Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa.

Bacteria and many non-metazoan Eukaryotes respond to stresses and threats using two-component systems (TCSs) comprising sensor kinases (SKs) and response regulators (RRs). Multikinase networks, where multiple SKs work together, detect and integrate different signals to control important lifestyle decisions such as sporulation and virulence. Here, we study interactions between two SKs from Pseudomonas aeruginosa, GacS and RetS, which control the switch between acute and chronic virulence. We demonstrate three mechanisms by which RetS attenuates GacS signalling: RetS takes phosphoryl groups from GacS-P; RetS has transmitter phosphatase activity against the receiver domain of GacS-P; and RetS inhibits GacS autophosphorylation. These mechanisms play important roles in vivo and during infection, and exemplify an unprecedented degree of signal processing by SKs that may be exploited in other multikinase networks.

Tandem Amplification of the Staphylococcal Cassette Chromosome mec Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus.

Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) ß-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 µg/ml to 130 µg/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ∼10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 µg/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.

Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosa.

With an increase in cases of multidrug-resistant Pseudomonas aeruginosa, alternative and adjunct treatments are needed, leading to renewed interest in bacteriophage therapy. There have been few clinically relevant studies of phage therapy against chronic lung infections. Using a novel murine model that uses a natural respiratory inhalation route of infection, we show that phage therapy is an effective treatment against chronic P. aeruginosa lung infections. We also show efficacy against P. aeruginosa in a biofilm-associated cystic fibrosis lung-like environment. These studies demonstrate the potential for phage therapy in the treatment of established and recalcitrant chronic respiratory tract infections.

Redeploying ß-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that ß-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.

Airborne dust and high temperatures are risk factors for invasive bacterial disease.

BACKGROUND: The Sahel region of West Africa has the highest bacterial meningitis attack and case fatality rate in the world. The effect of climatic factors on patterns of invasive respiratory bacterial disease is not well documented. OBJECTIVE: We aimed to assess the link between climatic factors and occurrence of invasive respiratory bacterial disease in a Sahel region of Niger. METHODS: We conducted daily disease surveillance and climatic monitoring over an 8-year period between January 1, 2003, and December 31, 2010, in Niamey, Niger, to determine risk factors for bacterial meningitis and invasive bacterial disease. We investigated the mechanistic effects of these factors on Streptococcus pneumoniae infection in mice. RESULTS: High temperatures and low visibility (resulting from high concentrations of airborne dust) were identified as significant risk factors for bacterial meningitis. Dust inhalation or exposure to high temperatures promoted progression of stable asymptomatic pneumococcal nasopharyngeal carriage to pneumonia and invasive disease. Dust exposure significantly reduced phagocyte-mediated bacterial killing, and exposure to high temperatures increased release of the key pneumococcal toxin pneumolysin through increased bacterial autolysis. CONCLUSION: Our findings show that climatic factors can have a substantial influence on infectious disease patterns, altering density of pneumococcal nasopharyngeal carriage, reducing phagocytic killing, and resulting in increased inflammation and tissue damage and consequent invasiveness. Climatic surveillance should be used to forecast invasive bacterial disease epidemics, and simple control measures to reduce particulate inhalation might reduce the incidence of invasive bacterial disease in regions of the world exposed to high temperatures and increased airborne dust.

AraC-Type Regulator Rbf Controls the Staphylococcus epidermidis Biofilm Phenotype by Negatively Regulating the icaADBC Repressor SarR.

Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. IMPORTANCE: The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating expression of sarR, which encodes an ica operon repressor. Furthermore, Rbf indirectly represses the ica operon activator, SarX. The data reveal complicated interplay between Rbf and two Sar family proteins in fine-tuning regulation of the biofilm phenotype and indicate that in the hierarchy of biofilm regulators, IcaR is dominant over the Rbf-SarR-SarX axis.

The major autolysin is redundant for Staphylococcus aureus USA300 LAC JE2 virulence in a murine device-related infection model.

The major Staphylococcus aureus autolysin, Atl, has been implicated in attachment to surfaces and release of extracellular DNA during biofilm formation under laboratory conditions. Consistent with this, polyclonal antibodies to the amidase and glucosaminidase domains of Atl inhibited in vitro biofilm formation. However, in a murine model of device-related infection the community-associated S. aureus strain USA300 LAC JE2 established a successful infection in the absence of atl These data indicate that Atl activity is not required for biofilm production in this infection model and reveal the importance of characterizing the contribution of biofilm phenotypes to virulence under in vivo conditions.

Role for the A domain of unprocessed accumulation-associated protein (Aap) in the attachment phase of the Staphylococcus epidermidis biofilm phenotype.

The polysaccharide intercellular adhesin or the cell wall-anchored accumulation-associated protein (Aap) mediates cellular accumulation during Staphylococcus epidermidis biofilm maturation. Mutation of sortase, which anchors up to 11 proteins (including Aap) to the cell wall, blocked biofilm development by the cerebrospinal fluid isolate CSF41498. Aap was implicated in this phenotype when Western blots and two-dimensional (2D) electrophoresis revealed increased levels of the protein in culture supernatants. Unexpectedly, reduced levels of primary attachment were associated with impaired biofilm formation by CSF41498 srtA and aap mutants. In contrast to previous studies, which implicated Aap proteolytic cleavage and, specifically, the Aap B domains in biofilm accumulation, the CSF41498 Aap protein was unprocessed. Furthermore, aap appeared to play a less important role in the biofilm phenotype of S. epidermidis 1457, in which the Aap protein is processed. Anti-Aap A-domain IgG inhibited primary attachment and biofilm formation in strain CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains CSF41498 and 1457 were identical, implicating altered protease activity in the differential Aap processing results in the two strains. These data reveal a new role for the A domain of unprocessed Aap in the attachment phase of biofilm formation and suggest that extracellular protease activity can influence whether Aap contributes to the attachment or accumulation phases of the S. epidermidis biofilm phenotype.

Rapid quantitative and qualitative analysis of biofilm production by Staphylococcus epidermidis under static growth conditions.

Rapid screening of biofilm forming capacity by Staphylococcus epidermidis is possible using in vitro assays with 96-well plates. This method first developed by Christensen et al. in 1985 is fast and does not require specialized instruments. Thus, laboratories with standard microbiology infrastructure and a 96-well plate reader can easily use this technique to generate data on the biofilm phenotypes of multiple S. epidermidis strains and clinical isolates. Furthermore, this method can be adapted to gain insights into biofilm regulation and the characteristics of biofilms produced by different S. epidermidis isolates. Although this assay is extremely useful for showing whether individual strains are biofilm-positive or biofilm-negative and distinguishing between form weak, moderate or strong biofilm, it is important to acknowledge that the absolute levels of biofilm produced by an individual strain can vary significantly between experiments meaning that strict adherence to the protocol used is of paramount importance. Furthermore, measuring biofilm under static conditions does not generally reflect in vivo conditions in which bacteria are often subjected to shear stresses under flow conditions. Hence, the biofilm characteristics of some strains are dramatically different under flow and static conditions. Nevertheless, rapid measurement of biofilm production under static conditions is a useful tool in the analysis of the S. epidermidis biofilm phenotype.

Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus device-associated infections.

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to ß-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to ß-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant Staphylococcus aureus by interfering with the agr quorum sensing system.

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.

Essential role for the major autolysin in the fibronectin-binding protein-mediated Staphylococcus aureus biofilm phenotype.

Staphylococcus aureus clinical isolates are capable of producing at least two distinct types of biofilm mediated by the fibronectin-binding proteins (FnBPs) or the icaADBC-encoded polysaccharide intercellular adhesin (PIA). Deletion of the major autolysin gene atl reduced primary attachment rates and impaired FnBP-dependent biofilm production on hydrophilic polystyrene in 12 clinical methicillin-resistant S. aureus (MRSA) isolates but had no effect on PIA-dependent biofilm production by 9 methicillin-susceptible S. aureus (MSSA) isolates. In contrast, Atl was required for both FnBP- and PIA-mediated biofilm development on hydrophobic polystyrene. Here we investigated the role of Atl in biofilm production on hydrophilic polystyrene. The alternative sigma factor σ(B), which represses RNAIII expression and extracellular protease production, was required for FnBP- but not PIA-dependent biofilm development. Furthermore, mutation of the agr locus enhanced FnBP-dependent biofilm development, whereas a sarA mutation, which increases protease production, blocked FnBP-mediated biofilm development. Mutation of sigB in MRSA isolate BH1CC lowered primary attachment rates, in part via reduced atl transcription. Posttranslational activation or inhibition of Atl activity with phenylmethylsulfonyl fluoride and polyanethole sodium sulfonate or mutation of the Atl amidase active site interfered with lytic activity and biofilm development. Consistent with these observations, extracellular DNA was important for the early stages of Atl/FnBP-dependent biofilm development. Further analysis of atl regulation revealed that atlR encodes a transcriptional repressor of the major autolysin and that an atlR::Tc(r) mutation in BH1CC enhanced biofilm-forming capacity. These data reveal an essential role for the major autolysin in the early events of the FnBP-dependent S. aureus biofilm phenotype.