Bacterial efflux pumps excrete SYTO™ dyes and lead to false-negative staining results.

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO™ dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO™ dyes should be used with caution on live samples.

Efficacy of rifampicin combination therapy against MRSA prosthetic vascular graft infections in a rat model.

Staphylococcus aureus is a major cause of prosthetic vascular graft or endograft infections (VGEIs) and the optimal choice of antibiotics is unclear. We investigated various antibiotic choices as either monotherapy or combination therapy with rifampicin against MRSA in vitro and in vivo. Fosfomycin, daptomycin and vancomycin alone or in combination with rifampicin was used against MRSA USA300 FPR3757. Each antibiotic was tested for synergism or antagonism with rifampicin in vitro, and all antibiotic regimens were tested against actively growing bacteria in media and non-growing bacteria in buffer, both as planktonic cells and in biofilms. A rat model of VGEI was used to quantify the therapeutic efficacy of antibiotics in vivo by measuring bacterial load on grafts and in spleen, liver and kidneys. In vitro, rifampicin combinations did not reveal any synergism or antagonism in relation to growth inhibition. However, quantification of bactericidal activity revealed a strong antagonistic effect, both on biofilms and planktonic cells. This effect was only observed when treating active bacteria, as all antibiotics had little or no effect on inactive cells. Only daptomycin showed some biocidal activity against inactive cells. In vivo evaluation of therapy against VGEI contrasted the in vitro results. Rifampicin significantly increased the efficacy of both daptomycin and vancomycin. The combination of daptomycin and rifampicin was by far the most effective, curing 8 of 13 infected animals. Our study demonstrates that daptomycin in combination with rifampicin shows promising potential against VGEI caused by MRSA. Furthermore, we show how in vitro evaluation of antibiotic combinations in laboratory media does not predict their therapeutic effect against VGEI in vivo, presumably due to a difference in the metabolic state of the bacteria.

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Considerations on the use of microsensors to profile dissolved H2 concentrations in microbial electrochemical reactors.

Measuring the distribution and dynamics of H2 in microbial electrochemical reactors is valuable to gain insights into the processes behind novel bioelectrochemical technologies, such as microbial electrosynthesis. Here, a microsensor method to measure and profile dissolved H2 concentrations in standard H-cell reactors is described. Graphite cathodes were oriented horizontally to enable the use of a motorized microprofiling system and a stereomicroscope was used to place the H2 microsensor precisely on the cathode surface. Profiling was performed towards the gas-liquid interface, while preserving the electric connections and flushing the headspace (to maintain anoxic conditions) and under strict temperature control (to overcome the temperature sensitivity of the microsensors). This method was tested by profiling six reactors, with and without inoculation of the acetogen Sporomusa ovata, at three different time points. H2 accumulated over time in the abiotic controls, while S. ovata maintained low H2 concentrations throughout the liquid phase (< 4 µM) during the whole experimental period. These results demonstrate that this setup generated insightful H2 profiles. However, various limitations of this microsensor method were identified, as headspace flushing lowered the dissolved H2 concentrations over time. Moreover, microsensors can likely not accurately measure H2 in the immediate vicinity of the solid cathode, because the solids cathode surface obstructs H2 diffusion into the microsensor. Finally, the reactors had to be discarded after microsensor profiling. Interested users should bear these considerations in mind when applying microsensors to characterize microbial electrochemical reactors.

The Effect of Enzymatic Treatment with Mutanase, Beta-Glucanase, and DNase on a Saliva-Derived Biofilm Model.

INTRODUCTION: The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase, and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model. METHODS: Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides. RESULTS: The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0% ± 1.3% SD vs. 36.9% ± 21.9% SD and 48.2% ± 14.9% SD). For biofilm removal, significant reductions of up to 73.2% ± 5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes. CONCLUSION: Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.

A New Device for In Situ Dental Biofilm Collection Additively Manufactured by Direct Metal Laser Sintering and Vat Photopolymerization.

Dental biofilms are complex medical biofilms that cause caries, the most prevalent disease of humankind. They are typically collected using handcrafted intraoral devices with mounted carriers for biofilm growth. As the geometry of handcrafted devices is not standardized, the shear forces acting on the biofilms and the access to salivary nutrients differ between carriers. The resulting variability in biofilm growth renders the comparison of different treatment modalities difficult. The aim of the present work was to design and validate an additively manufactured intraoral device with a dental bar produced by direct metal laser sintering and vat photopolymerized inserts with standardized geometry for the mounting of biofilm carriers. Additive manufacturing reduced the production time and cost, guaranteed an accurate fit of the devices and facilitated the handling of carriers without disturbing the biofilm. Biofilm growth was robust, with increasing thickness over time and moderate inter- and intraindividual variation (coefficients of variance 0.48-0.87). The biofilms showed the typical architecture and composition of dental biofilms, as evidenced by confocal microscopy and 16S rRNA gene sequencing. Deeper inserts offering increased protection from shear tended to increase the biofilm thickness, whereas prolonged exposure to sucrose during growth increased the biofilm volume but not the thickness. Ratiometric pH imaging revealed considerable pH variation between participants and also inside single biofilms. Intraoral devices for biofilm collection constitute a new application for medical additive manufacturing and offer the best possible basis for studying the influence of different treatment modalities on biofilm growth, composition, and virulence. The Clinical Trial Registration number is: 1-10-72-193-20.

Fibrinolytic and antibiotic treatment of prosthetic vascular graft infections in a novel rat model.

OBJECTIVES: We developed a rat model of prosthetic vascular graft infection to assess, whether the fibrinolytic tissue plasminogen activator (tPA) could increase the efficacy of antibiotic therapy. MATERIALS AND METHODS: Rats were implanted a polyethylene graft in the common carotid artery, pre-inoculated with approx. 6 log10 colony forming units (CFU) of methicillin resistant Staphylococcus aureus. Ten days after surgery, rats were randomized to either: 0.9% NaCl (n = 8), vancomycin (n = 8), vancomycin + tPA (n = 8), vancomycin + rifampicin (n = 18) or vancomycin + rifampicin + tPA (n = 18). Treatment duration was seven days. Approximately 36 hours after the end of treatment, the rats were euthanized, and grafts and organs were harvested for CFU enumeration. RESULTS: All animals in the control group had significantly higher CFU at the time of euthanization compared to bacterial load found on the grafts prior to inoculation (6.45 vs. 4.36 mean log10 CFU/mL, p = 0.0011), and both the procedure and infection were well tolerated. Vancomycin and rifampicin treatment were superior to monotherapy with vancomycin, as it lead to a marked decrease in median bacterial load on the grafts (3.50 vs. 6.56 log10 CFU/mL, p = 0.0016). The addition of tPA to vancomycin and rifampicin combination treatment did not show a further decrease in bacterial load (4.078 vs. 3.50 log10 CFU/mL, p = 0.26). The cure rate was 16% in the vancomycin + rifampicin group vs. 37.5% cure rate in the vancomycin + rifampicin + tPA group. Whilst interesting, this trend was not significant at our sample size (p = 0.24). CONCLUSION: We developed the first functional model of an arterial prosthetic vascular graft infection in rats. Antibiotic combination therapy with vancomycin and rifampicin was superior to vancomycin monotherapy, and the addition of tPA did not significantly reduce bacterial load, nor significantly increase cure rate.

Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells.

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

Polymyxin B complexation enhances the antimicrobial potential of graphene oxide.

Introduction: The antibacterial activity of graphene oxide (GO) has been widely explored and tested against various pathogenic bacterial strains. Although antimicrobial activity of GO against planktonic bacterial cells was demonstrated, its bacteriostatic and bactericidal effect alone is not sufficient to damage sedentary and well protected bacterial cells inside biofilms. Thus, to be utilized as an effective antibacterial agent, it is necessary to improve the antibacterial activity of GO either by integration with other nanomaterials or by attachment of antimicrobial agents. In this study, antimicrobial peptide polymyxin B (PMB) was adsorbed onto the surface of pristine GO and GO functionalized with triethylene glycol. Methods: The antibacterial effects of the resulting materials were examined by evaluating minimum inhibitory concentration, minimum bactericidal concentration, time kill assay, live/dead viability staining and scanning electron microscopy. Results and discussion: PMB adsorption significantly enhanced the bacteriostatic and bactericidal activity of GO against both planktonic cells and bacterial cells in biofilms. Furthermore, the coatings of PMB-adsorbed GO applied to catheter tubes strongly mitigated biofilm formation, by preventing bacterial adhesion and killing the bacterial cells that managed to attach. The presented results suggest that antibacterial peptide absorption can significantly enhance the antibacterial activity of GO and the resulting material can be effectively used not only against planktonic bacteria but also against infectious biofilms.

Biofilm formation and inflammatory potential of Staphylococcus saccharolyticus: A possible cause of orthopedic implant-associated infections.

Staphylococcus saccharolyticus, a coagulase-negative staphylococcal species, has some unusual characteristics for human-associated staphylococci, such as slow growth and its preference for anoxic culture conditions. This species is a relatively abundant member of the human skin microbiota, but its microbiological properties, as well as the pathogenic potential, have scarcely been investigated so far, despite being occasionally isolated from different types of infections including orthopedic implant-associated infections. Here, we investigated the growth and biofilm properties of clinical isolates of S. saccharolyticus and determined host cell responses. Growth assessments in anoxic and oxic conditions revealed strain-dependent outcomes, as some strains can also grow aerobically. All tested strains of S. saccharolyticus were able to form biofilm in a microtiter plate assay. Strain-dependent differences were determined by optical coherence tomography, revealing that medium supplementation with glucose and sodium chloride enhanced biofilm formation. Visualization of the biofilm by confocal laser scanning microscopy revealed the role of extracellular DNA in the biofilm structure. In addition to attached biofilms, S. saccharolyticus also formed bacterial aggregates at an early stage of growth. Transcriptome analysis of biofilm-grown versus planktonic cells revealed a set of upregulated genes in biofilm-embedded cells, including factors involved in adhesion, colonization, and competition such as epidermin, type I toxin-antitoxin system, and phenol-soluble modulins (beta and epsilon). To investigate consequences for the host after encountering S. saccharolyticus, cytokine profiling and host cell viability were assessed by infection experiments with differentiated THP-1 cells. The microorganism strongly triggered the secretion of the tested pro-inflammatory cyto- and chemokines IL-6, IL-8, and TNF-alpha, determined at 24 h post-infection. S. saccharolyticus was less cytotoxic than Staphylococcus aureus. Taken together, the results indicate that S. saccharolyticus has substantial pathogenic potential. Thus, it can be a potential cause of orthopedic implant-associated infections and other types of deep-seated infections.

JMM Profile: Staphylococcus epidermidis.

Staphylococcus epidermidis is the most abundant commensal bacterium of human skin. Despite protecting us from foreign invaders, S. epidermidis itself exploits human vulnerability when given the opportunity. Such opportunities arise when patients are immunocompromised or when biomedical implants present an opportunity to colonize the surface and form biofilms. S. epidermidis is one of the most frequently isolated organisms from implanted devices and from bloodstream infections. However, S. epidermidis infections are often recalcitrant to antibiotics because of biofilm-associated antibiotic tolerance. Furthermore, the emergence and spread of nearly pan-resistant strains is a considerable health concern. Symptoms can be subclinical, making diagnosis challenging, and treatment with antibiotics is inefficient. For now, infection prevention remains the best strategy available.

The giant staphylococcal protein Embp facilitates colonization of surfaces through Velcro-like attachment to fibrillated fibronectin.

Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, extracellular matrix-binding protein (Embp), which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multivalent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether.

A usually harmless bacterium called Staphylococcus epidermidis lives on human skin. Sometimes it makes its way into the bloodstream through a cut or surgical procedure, but it rarely causes blood infections. It can, however, cause severe infections when it attaches to the surface of a medical implant like a pacemaker or an artificial replacement joint. It does this by forming a colony of bacteria on the implant's surface called a biofilm, which protects the bacteria from destruction by the immune system or antibiotics. Understanding how Staphylococcus epidermidis implant infections start is critical to preventing them. This information may help scientists develop infection-resistant implants or new treatments for implant infections. Scientists suspect that Staphylococcus epidermidis attaches to implants by binding to a human protein called fibronectin, which coats medical implants in the human body. Another protein on the surface of the bacteria, called Embp, facilitates the connection. But why the bacteria attach to fibronectin on implants, and not fibronectin molecules in the bloodstream, is unclear. Now, Khan, Aslan et al. show that Embp forms a Velcro-like bond with fibronectin on the surface of implants. In the experiments, Khan and Aslan et al. used powerful microscopes to create 3-dimensional images of the interactions between Embp and fibronectin. The experiments showed that Embp's attachment site is hidden on the globe-shaped form of fibronectin circulating in the blood. But when fibronectin covers an implant surface, it forms a fibrous network, and Embp can attach to it with up to 50 Velcro-like individual connections. These many weak connections form a strong bond that withstands the force of blood pumping past. The experiments show that the fibrous coating of fibronectin on implants makes them a hotspot for Staphylococcus epidermidis infections. Finding ways to block Embp from attaching to fibronectin on implants, or altering the form fibronectin takes on implants, may help prevent these infections. Many bacteria that form biofilms have an Embp-like protein. As a result, these discoveries may also help scientists develop prevention or treatment strategies for other bacterial biofilm infections.

The Bacterial Life Cycle in Textiles is Governed by Fiber Hydrophobicity.

Colonization of textiles and subsequent metabolic degradation of sweat and sebum components by axillary skin bacteria cause the characteristic sweat malodor and discoloring of dirty clothes. Once inside the textile, the bacteria can form biofilms that are hard to remove by conventional washing. When the biofilm persists after washing, the textiles retain the sweat odor. To design biofilm removal and prevention strategies, the bacterial behavior needs to be understood in depth. Here, we aim to study the bacterial behavior in each of the four stages of the bacterial life cycle in textiles: adhesion, growth, drying, and washing. To accomplish this, we designed a novel in vitro model to mimic physiological sweating in cotton and polyester textiles, in which many of the parameters that influence bacterial behavior could be controlled. Due to the higher hydrophobicity, polyester adhered more bacteria and absorbed more sebum, the bacteria's primary nutrient source. Bacteria were therefore also more active in polyester textiles. However, polyester did not bind water as well as cotton. The increased water content of cotton allowed some species to retain a higher activity after the textile had dried. However, none of the textiles retained enough water upon drying to prevent the bacteria from adhering irreversibly to the textile fibers. This work demonstrates that bacterial colonization of textiles depends partially on the hydrophobic and hygroscopic properties of the textile material, indicating that it might be possible to direct bacterial behavior in a more favorable direction by modifying these surface properties. IMPORTANCE During sweating, bacteria from the skin enter the worn textile along with the sweat. Once inside the clothes, the bacteria produce sweat malodor and form colonies that are extremely hard to remove by washing. Over time, this leads to a decreasing textile quality and consumer comfort. To design prevention and removal mechanisms, we investigated the behavior of bacteria during the four stages of their life cycle in textiles: adhesion, growth, drying, and washing. The bacterial behavior in textiles during all four stages is found to be affected by the textile's ability to bind water and fat. The study indicates that sweat malodor and bacterial accumulation in textiles over time can be reduced by making the textiles more repellant to water and fat.

Human Fibrinogen Inhibits Amyloid Assembly of Most Phenol-Soluble Modulins from Staphylococcus aureus.

Functional amyloids are highly organized protein/peptide structures that inter alia promote biofilm formation in different bacteria. One such example is provided by a family of 20-45 residue-long peptides called phenol-soluble modulins (PSMs) from Staphylococcus aureus. External components such as eukaryotic host proteins, which alter self-assembly of bacterial amyloids, can affect the biofilm matrix. Here, we studied the effect of the highly prevalent human plasma protein fibrinogen (Fg) on fibrillation of PSMs. Fg inhibits or suppresses fibrillation of most PSMs tested (PSMα1, PSMß1, and PSMß2) except for PSMα3, whose already rapid aggregation is accelerated even further by Fg but leads to amorphous ß-rich aggregates rather than fibrils. Fg also induces PSMß2 to form amorphous aggregates and diverts PSMα1 into off-pathway oligomers which consist of both Fg and PSMα1 and cannot seed fibrillation. Peptide arrays showed that Fg bound to the N-terminus of PSMα1, while it bound to the entire length of PSMα3 (except the C terminus) and to the C-termini of PSMß1 and PSMß2. The latter peptides are all positively charged, while Fg is negatively charged at physiological pH. The positive charges complement Fg's net negative charge of -7.6 at pH 7.4. Fg's ability to inhibit PSM fibrillation reveals a potential host-defense mechanism to prevent bacterial biofilm growth and infections in the human body.

Management of oral biofilms by nisin delivery in adhesive microdevices.

Numerous beneficial microbes thrive in the oral cavity where they form biofilms on dental and mucosal surfaces to get access to nutrients, and to avoid being carried away with the saliva. However, biofilm formation is also a virulence factor as it also protects pathogenic bacteria, providing them with an environment for proliferation causing oral infections. Oral hygiene relies on mechanical removal of biofilms. Some oral care products also contain antimicrobials, but effective eradication of biofilms with antimicrobials requires both a high concentration and long exposure time. In the present communication, we investigate the potential of using miniaturized drug delivery devices, known as microcontainers (MCs), to deliver the antimicrobial peptide, nisin to an oral multi-species biofilm. MCs are loaded with nisin and X-ray micro-computed tomography reveals a full release of nisin through a chitosan lid within 15 min. Chitosan-coated MCs display substantial bioadhesion to the buccal mucosa compared to non-coated MCs (68.6 ± 14.3% vs 33.8 ± 5.2%). Confocal monitoring of multi-species biofilms reveals antibacterial effects of nisin-loaded chitosan-coated MCs with a faster onset (after 3 h) compared to solution-based delivery (after 9 h). Our study shows the potential of using MCs for treatment of multi-species oral biofilms and is encouraging for further design of drug delivery devices to treat oral diseases.

Genome Sequence of Staphylococcus epidermidis AUH4567, a Clinical Isolate from an Infected Central Venous Catheter.

Staphylococcus epidermidis is a common cause of implant-associated infections, and this is related to its ability to form biofilms. Strain-to-strain variability in biofilm formation is likely caused by genetic differences. Here, we present a draft genome of S. epidermidis AUH4567, which was isolated from a central venous catheter infection.

Activation of the Two-Component System LisRK Promotes Cell Adhesion and High Ampicillin Tolerance in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen which can survive in harsh environmental conditions. It responds to external stimuli through an array of two-component systems (TCS) that sense external cues. Several TCS, including LisRK, have been linked to Listeria's ability to grow at slightly elevated antibiotic levels. The aim of this study was to determine if the TCS LisRK is also involved in acquiring the high antibiotic tolerance that is characteristic of persister cells. LisRK activates a response that leads to remodeling of the cell envelope, and we therefore hypothesized that activation of LisRK could also increase in the cells' adhesiveness and initiate the first step in biofilm formation. We used a ΔlisR mutant to study antibiotic tolerance in the presence and absence of LisRK, and a GFP reporter strain to visualize the activation of LisRK in L. monocytogenes LO28 at a single-cell level. LisRK was activated in most cells in stationary phase cultures. Antimicrobial susceptibility tests showed that LisRK was required for the generation of ampicillin tolerance under these conditions. The wildtype strain tolerated exposure to ampicillin at 1,000 × inhibitory levels for 24 h, and the fraction of surviving cells was 20,000-fold higher in the wildtype strain compared to the ΔlisR mutant. The same protection was not offered to other antibiotics (vancomycin, gentamicin, tetracycline), and the mechanism for antibiotic tolerance is thus highly specific. Furthermore, quantification of bacterial attachment rates and attachment force also revealed that the absence of a functional LisRK rendered the cells less adhesive. Hence, LisRK TCS promotes multiple protective mechanisms simultaneously.

Phenol-Soluble Modulins Modulate Persister Cell Formation in Staphylococcus aureus.

Staphylococcus aureus is a human pathogen that can cause chronic and recurrent infections and is recalcitrant to antibiotic chemotherapy. This trait is partly attributed to its ability to form persister cells, which are subpopulations of cells that are tolerant to lethal concentrations of antibiotics. Recently, we showed that the phenol-soluble modulins (PSMs) expressed by S. aureus reduce persister cell formation. PSMs are a versatile group of toxins that, in addition to toxicity, form amyloid-like fibrils thought to support biofilm structures. Here, we examined individual or combined synthetic PSMα peptides and their equivalent amyloid-like fibrils on ciprofloxacin-selected S. aureus persister cells. We found that PSMα2 and the mixture of all four alpha peptides consistently were able to reduce persister frequency in all growth phases, and this activity was specifically linked to the presence of the soluble peptide as no effect was seen with fibrillated peptides. Persister reduction was particularly striking in a mutant that, due to mutations in the Krebs cycle, has enhanced ability to form persisters with PSMα4 and the combination of peptides being most effective. In biofilms, only the combination of peptides displayed persister reducing activity. Collectively, we report the individual contributions of PSMα peptides to persister cell reduction and that the combination of peptides generally was most effective. Strikingly, the fibrillated peptides lost activity and thus, if formed in bacterial cultures, they will be inactive against persister cells. Further studies will be needed to address the biological role of phenol-soluble modulins in reducing persister cells.

Antifouling properties of layer by layer DNA coatings.

Fouling is a major concern for solid/liquid interfaces of materials used in different applications. One approach of fouling control is the use of hydrophilic polymer coatings made from poly-anions and poly-cations using the layer-by-layer (LBL) method. The authors hypothesized that the poly-anionic properties and the poly-phosphate backbone of DNA would provide anti-biofouling and anti-scaling properties. To this end, poly(ethyleneimine)/DNA LBL coatings against microbial and inorganic fouling were developed, characterized and evaluated. DNA LBL coatings reduced inorganic fouling from tap water by 90% when incubated statically or under flow conditions mimicking surfaces in heat exchangers. The coatings also impaired biofilm formation by 93% on stainless steel from tap water, and resulted in a 97% lower adhesion force and reduced initial attachment of the human pathogens Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa on glass. This study demonstrates a proof of concept that LBL coatings with poly-anions harboring phosphate groups can address fouling in several applications.