Pseudomonas aeruginosa Increases the Sensitivity of Biofilm-Grown Staphylococcus aureus to Membrane-Targeting Antiseptics and Antibiotics.
ABSTRACT: Pseudomonas aeruginosa and Staphylococcus aureus often cause chronic, recalcitrant infections in large part due to their ability to form biofilms. The biofilm mode of growth enables these organisms to withstand antibacterial insults that would effectively eliminate their planktonic counterparts. We found that P. aeruginosa supernatant increased the sensitivity of S. aureus biofilms to multiple antimicrobial compounds, including fluoroquinolones and membrane-targeting antibacterial agents, including the antiseptic chloroxylenol. Treatment of S. aureus with the antiseptic chloroxylenol alone did not decrease biofilm cell viability; however, the combination of chloroxylenol and P. aeruginosa supernatant led to a 4-log reduction in S. aureus biofilm viability compared to exposure to chloroxylenol alone. We found that the P. aeruginosa-produced small molecule 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) is responsible for the observed heightened sensitivity of S. aureus to chloroxylenol. Similarly, HQNO increased the susceptibility of S. aureus biofilms to other compounds, including both traditional and nontraditional antibiotics, which permeabilize bacterial membranes. Genetic and phenotypic studies support a model whereby HQNO causes an increase in S. aureus membrane fluidity, thereby improving the efficacy of membrane-targeting antiseptics and antibiotics. Importantly, our data show that P. aeruginosa exoproducts can enhance the ability of various antimicrobial agents to kill biofilm populations of S. aureus that are typically difficult to eradicate. Finally, our discovery that altering membrane fluidity shifts antimicrobial sensitivity profiles of bacterial biofilms may guide new approaches to target persistent infections, such as those commonly found in respiratory tract infections and in chronic wounds.IMPORTANCE The thick mucus in the airways of cystic fibrosis (CF) patients predisposes them to frequent, polymicrobial respiratory infections. Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from the airways of individuals with CF, as well as from diabetic foot ulcers and other wounds. Both organisms form biofilms, which are notoriously difficult to eradicate and promote chronic infection. In this study, we have shown that P. aeruginosa-secreted factors can increase the efficacy of compounds that alone have little or no bactericidal activity against S. aureus biofilms. In particular, we discovered that P. aeruginosa exoproducts can potentiate the antistaphylococcal activity of phenol-based antiseptics and other membrane-active drugs. Our findings illustrate that polymicrobial interactions can dramatically increase antibacterial efficacy in vitro and suggest that altering membrane physiology promotes the ability of certain drugs to kill bacterial biofilms-knowledge that may provide a path for the discovery of new biofilm-targeting antimicrobial strategies.
Project description:The airways of cystic fibrosis (CF) patients have thick mucus, which fosters chronic, polymicrobial infections. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients. In this study, we tested whether P. aeruginosa influences the susceptibility of S. aureus to frontline antibiotics used to treat CF lung infections. Using our in vitro coculture model, we observed that addition of P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic significantly decreased the susceptibility of S. aureus to vancomycin. Mutant analyses showed that 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), a component of the P. aeruginosa Pseudomonas quinolone signal (PQS) system, protects S. aureus from the antimicrobial activity of vancomycin. Similarly, the siderophores pyoverdine and pyochelin also contribute to the ability of P. aeruginosa to protect S. aureus from vancomycin, as did growth under anoxia. Under our experimental conditions, HQNO, P. aeruginosa supernatant, and growth under anoxia decreased S. aureus growth, likely explaining why this cell wall-targeting antibiotic is less effective. P. aeruginosa supernatant did not confer additional protection to slow-growing S. aureus small colony variants. Importantly, P. aeruginosa supernatant protects S. aureus from other inhibitors of cell wall synthesis as well as protein synthesis-targeting antibiotics in an HQNO- and siderophore-dependent manner. We propose a model whereby P. aeruginosa causes S. aureus to shift to fermentative growth when these organisms are grown in coculture, leading to reduction in S. aureus growth and decreased susceptibility to antibiotics targeting cell wall and protein synthesis.IMPORTANCE Cystic fibrosis (CF) lung infections are chronic and difficult to eradicate. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients and are associated with poor patient outcomes. Both organisms adopt a biofilm mode of growth, which contributes to high tolerance to antibiotic treatment and the recalcitrant nature of these infections. Here, we show that P. aeruginosa exoproducts decrease the sensitivity of S. aureus biofilm and planktonic populations to vancomycin, a frontline antibiotic used to treat methicillin-resistant S. aureus in CF patients. P. aeruginosa also protects S. aureus from other cell wall-active antibiotics as well as various classes of protein synthesis inhibitors. Thus, interspecies interactions can have dramatic and unexpected consequences on antibiotic sensitivity. This study underscores the potential impact of interspecies interactions on antibiotic efficacy in the context of complex, polymicrobial infections.
Project description:<h4>Background</h4>Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.<h4>Results</h4>We demonstrated that HQNO stimulates S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the alpha-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background.<h4>Conclusions</h4>These results suggest that S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.
Project description:Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent pathogens in airway infections of cystic fibrosis (CF) patients. We studied how these pathogens coexist and interact with each other. Clinical isolates of both species were retrieved from adult CF patients. Culture supernatants from 63 P. aeruginosa isolates triggered a wide range of biofilm-stimulatory activities when added to the culture of a control S. aureus strain. The extent of biofilm formation by S. aureus was positively correlated to the levels of the 2-alkyl-4-(1H)-quinolones (AQs) Pseudomonas Quinolone Signal (PQS) and 2-heptyl-4-hydroxy quinoline N-oxide (HQNO) produced by the P. aeruginosa isolates. Supernatants from P. aeruginosa isogenic mutants deficient in PQS and HQNO production stimulated significantly less biofilm formation by S. aureus than that seen with the parental strain PA14. When studying co-isolated pairs of P. aeruginosa and S. aureus retrieved from patients showing both pathogens, P. aeruginosa supernatants stimulated less biofilm production by the S. aureus counterparts compared to that observed using the control S. aureus strain. Accordingly, some P. aeruginosa isolates produced low levels of exoproducts and also some of the clinical S. aureus isolates were not stimulated by their co-isolates or by PA14 despite adequate production of HQNO. This suggests that colonization of the CF lungs promotes some type of strain selection, or that co-existence requires specific adaptations by either or both pathogens. Results provide insights on bacterial interactions in CF.
Project description:Infection of intravascular catheters by Staphylococcus aureus is a significant risk factor within the health care setting. To treat these infections and attempt salvage of an intravascular catheter, antimicrobial lock solutions (ALSs) are being increasingly used. However, the most effective ALSs against these biofilm-mediated infections have yet to be determined, and clinical practice varies greatly. The purpose of this study was to evaluate and compare the efficacies of antibiotics and antiseptics in current clinical use against biofilms produced by reference and clinical isolates of S. aureus Static and flow biofilm assays were developed using newly described in vivo-relevant conditions to examine the effect of each agent on S. aureus within the biofilm matrix. The antibiotics daptomycin, tigecycline, and rifampin and the antiseptics ethanol and Taurolock inactivated established S. aureus biofilms, while other commonly used antistaphylococcal antibiotics and antiseptic agents were less effective. These findings were confirmed by live/dead staining of S. aureus biofilms formed and treated within a flow cell model. The results from this study demonstrate the most effective clinically used agents and their concentrations which should be used within an ALS to treat S. aureus-mediated intravascular catheter-related infections.
Project description:Antimicrobial resistance is a significant threat to the treatment of infectious disease. Multiple mechanisms of resistance to different classes of antibiotics have been identified and well-studied. However, these mechanisms are studied with bacteria in isolation, whereas often, infections have a polymicrobial basis. Using a biofilm slide chamber model, we visualized the formation and development of clinical <i>Pseudomonas aeruginosa</i> biofilms in the presence of secreted <i>Staphylococcus aureus</i> exoproducts, two bacteria that commonly co-infect pediatric patients with cystic fibrosis. We showed that, over time, certain isolates of <i>P. aeruginosa</i> can form different biofilm architecture in the presence of <i>S. aureus</i> exoproducts. We further determined that this interaction was dependent on Psl produced by <i>P. aeruginosa</i> and staphylococcal protein A from <i>S. aureus</i>. Importantly, we identified a mechanism of antibiotic resistance to tobramycin that is dependent on the polymicrobial interactions between these two bacteria. This interaction occurred in isolates of <i>P. aeruginosa</i> recovered from children with cystic fibrosis who failed to clear <i>P. aeruginosa</i> following inhaled tobramycin treatment.
Project description:Choline geranate (also described as Choline And GEranic acid, or CAGE) has been developed as a novel biocompatible antiseptic material capable of penetrating skin and aiding the transdermal delivery of co-administered antibiotics. The antibacterial properties of CAGE were analyzed against 24 and 72 hour old biofilms of 11 clinically isolated ESKAPE pathogens (defined as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp, respectively), including multidrug resistant (MDR) isolates. CAGE was observed to eradicate in vitro biofilms at concentrations as low as 3.56 mM (0.156% v:v) in as little as 2 hours, which represents both an improved potency and rate of biofilm eradication relative to that reported for most common standard-of-care topical antiseptics in current use. In vitro time-kill studies on 24 hour old Staphylococcus aureus biofilms indicate that CAGE exerts its antibacterial effect upon contact and a 0.1% v:v solution reduced biofilm viability by over three orders of magnitude (a 3log10 reduction) in 15 minutes. Furthermore, disruption of the protective layer of exopolymeric substances in mature biofilms of Staphylococcus aureus by CAGE (0.1% v:v) was observed in 120 minutes. Insight into the mechanism of action of CAGE was provided with molecular modeling studies alongside in vitro antibiofilm assays. The geranate ion and geranic acid components of CAGE are predicted to act in concert to integrate into bacterial membranes, affect membrane thinning and perturb membrane homeostasis. Taken together, our results show that CAGE demonstrates all properties required of an effective topical antiseptic and the data also provides insight into how its observed antibiofilm properties may manifest.
Project description:Infective complications are a major factor contributing to wound chronicity and can be associated with significant morbidity or mortality. Wound bacteria are protected in biofilm communities and are highly resistant to immune system components and to antimicrobials used in wound therapy. There is an urgent medical need to more effectively eradicate wound biofilm pathogens. In the present work, we tested the impact of such commonly used antibiotics and antiseptics as gentamycin, ciprofloxacin, octenidine, chlorhexidine, polihexanidine, and ethacridine lactate delivered to Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the presence of rotating magnetic fields (RMFs) of 10-50?Hz frequency and produced by a customized RMF generator. Fifty percent greater reduction in biofilm growth and biomass was observed after exposure to RMF as compared to biofilms not exposed to RMF. Our results suggest that RMF as an adjunct to antiseptic wound care can significantly improve antibiofilm activity, which has important translational potential for clinical applications.
Project description:BACKGROUND:Diabetic foot infections (DFIs) are a frequent complication of Diabetes mellitus and a major cause of nontraumatic limb amputations. The Gram-positive bacterium Staphylococcus aureus, known for its resilient biofilms and antibiotic resistant profile, is the most frequent DFI pathogen. It is urgent to develop innovative treatments for these infections, being the antimicrobial peptide (AMP) nisin a potential candidate. We have previously proposed the use of a guar gum biogel as a delivery system for nisin. Here, we evaluated the potential of the nisin-biogel to enhance the efficacy of conventional antibiotics and antiseptics against DFIs S. aureus clinical isolates. METHODS:A collection of 23 S. aureus strains isolated from DFI patients, including multidrug- and methicillin-resistant strains, was used. The antimicrobial activity of the nisin-biogel was tested alone and in different combinations with the antiseptic chlorhexidine and the antibiotics clindamycin, gentamicin and vancomycin. Isolates' in vitro susceptibility to the different protocols was assessed using broth microdilution methods in order to determine their ability to inhibit and/or eradicate established S. aureus biofilms. Antimicrobials were added to the 96-well plates every 8 h to simulate a typical DFI treatment protocol. Statistical analysis was conducted using RCBD ANOVA in SPSS. RESULTS:The nisin-biogel showed a high antibacterial activity against biofilms formed by DFI S. aureus. The combined protocol using nisin-biogel and chlorhexidine presented the highest efficacy in biofilm formation inhibition, significantly higher (p<0.05) than the ones presented by the antibiotics-based protocols tested. Regarding biofilm eradication, there were no significant differences (p>0.05) between the activity of the combination nisin-biogel plus chlorhexidine and the conventional antibiotic-based protocols. CONCLUSIONS:Results provide a valuable contribution for the development of complementary strategies to conventional antibiotics protocols. A combined protocol including chlorhexidine and nisin-biogel could be potentially applied in medical centres, contributing for the reduction of antibiotic administration, selection pressure on DFI pathogens and resistance strains dissemination.
Project description:Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner.IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ?30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.
Project description:The increasing incidence of non-healing wounds constitutes a pivotal socio-economic burden. 60-80% of chronic wounds are colonized by pathogenic microorganisms within a protective extracellular polymeric substance, bearing a great challenge in wound management. Human plasma was used to prepare the biofilm model (hpBIOM), adding pathogens to the plasma and forming Coagula-like discs with integrated pathogens were produced. The antiseptics Octenisept and Lavasorb were tested regarding their antibacterial properties on clinically relevant biofilm-growing bacteria (MRSA, P. aeruginosa) in the hpBIOM. Biofilm-typical glycocalyx-formation was confirmed using immunohistochemical staining. Treatment of a 12 h-maturated biofilm with Octenisept resulted in complete eradication of P. aeruginosa and MRSA after 48?h. Lavasorb proved less effective than Octenisept in this setting. In more mature biofilms (24?h), both antiseptics showed a delayed, partially decreased efficacy. Summarized, the hpBIOM provides essential factors for a translational research approach to be used for detailed human biofilm analyses and evaluation of antimicrobial/-biofilm properties of established and novel therapeutic strategies and products. Octenisept and Lavasorb showed an attenuated efficacy in the hpBIOM compared to planktonic conditions and previously published biofilm-studies, prompting the question for the necessity of introducing new international standards and pre-admission requirements on a translational base.