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Sample records for aeruginosa biofilm infections

  1. Pseudomonas aeruginosa forms Biofilms in Acute InfectionIndependent of Cell-to-Cell Signaling

    SciTech Connect

    Schaber, J. Andy; Triffo, W.J.; Suh, Sang J.; Oliver, Jeffrey W.; Hastert, Mary C.; Griswold, John A.; Auer, Manfred; Hamood, Abdul N.; Rumbaugh, Kendra P.

    2006-09-20

    Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 hours of infection in thermally-injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections. P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild type and QS-deficient P. aeruginosa formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independent of QS.

  2. Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy

    PubMed Central

    Gnanadhas, Divya Prakash; Elango, Monalisha; Datey, Akshay; Chakravortty, Dipshikha

    2015-01-01

    Bacterial biofilms are associated with 80–90% of infections. Within the biofilm, bacteria are refractile to antibiotics, requiring concentrations >1,000 times the minimum inhibitory concentration. Proteins, carbohydrates and DNA are the major components of biofilm matrix. Pseudomonas aeruginosa (PA) biofilms, which are majorly associated with chronic lung infection, contain extracellular DNA (eDNA) as a major component. Herein, we report for the first time that L-Methionine (L-Met) at 0.5 μM inhibits Pseudomonas aeruginosa (PA) biofilm formation and disassembles established PA biofilm by inducing DNase expression. Four DNase genes (sbcB, endA, eddB and recJ) were highly up-regulated upon L-Met treatment along with increased DNase activity in the culture supernatant. Since eDNA plays a major role in establishing and maintaining the PA biofilm, DNase activity is effective in disrupting the biofilm. Upon treatment with L-Met, the otherwise recalcitrant PA biofilm now shows susceptibility to ciprofloxacin. This was reflected in vivo, in the murine chronic PA lung infection model. Mice treated with L-Met responded better to antibiotic treatment, leading to enhanced survival as compared to mice treated with ciprofloxacin alone. These results clearly demonstrate that L-Met can be used along with antibiotic as an effective therapeutic against chronic PA biofilm infection. PMID:26521707

  3. OligoG CF-5/20 Disruption of Mucoid Pseudomonas aeruginosa Biofilm in a Murine Lung Infection Model.

    PubMed

    Hengzhuang, Wang; Song, Zhijun; Ciofu, Oana; Onsøyen, Edvar; Rye, Philip D; Høiby, Niels

    2016-05-01

    Biofilm growth is a universal survival strategy for bacteria, providing an effective and resilient approach for survival in an otherwise hostile environment. In the context of an infection, a biofilm provides resistance and tolerance to host immune defenses and antibiotics, allowing the biofilm population to survive and thrive under conditions that would destroy their planktonic counterparts. Therefore, the disruption of the biofilm is a key step in eradicating persistent bacterial infections, as seen in many types of chronic disease. In these studies, we used both in vitro minimum biofilm eradication concentration (MBEC) assays and an in vivo model of chronic biofilm infection to demonstrate the biofilm-disrupting effects of an alginate oligomer, OligoG CF-5/20. Biofilm infections were established in mice by tracheal instillation of a mucoid clinical isolate of Pseudomonas aeruginosa embedded in alginate polymer beads. The disruption of the biofilm by OligoG CF-5/20 was observed in a dose-dependent manner over 24 h, with up to a 2.5-log reduction in CFU in the infected mouse lungs. Furthermore, in vitro assays showed that 5% OligoG CF-5/20 significantly reduced the MBEC for colistin from 512 μg/ml to 4 μg/ml after 8 h. These findings support the potential for OligoG CF-5/20 as a biofilm disruption agent which may have clinical value in reducing the microbial burden in chronic biofilm infections. PMID:26833153

  4. Prevalence of Pseudomonas aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with chronic periodontal infection.

    PubMed

    Souto, Renata; Silva-Boghossian, Carina M; Colombo, Ana Paula Vieira

    2014-01-01

    P. aeruginosa and Acinetobacter spp. are important pathogens associated with late nosocomial pneumonia in hospitalized and institutionalized individuals. The oral cavity may be a major source of these respiratory pathogens, particularly in the presence of poor oral hygiene and periodontal infection. This study investigated the prevalence of P. aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with periodontal disease or health. Samples were obtained from 55 periodontally healthy (PH) and 169 chronic periodontitis (CP) patients. DNA was obtained from the samples and detection of P. aeruginosa and Acinetobacter spp. was carried out by multiplex and nested PCR. P. aeruginosa and Acinetobacter spp. were detected in 40% and 45% of all samples, respectively. No significant differences in the distribution of these microorganisms between men and women, subgingival biofilm and saliva samples, patients ≤ 35 and > 35 years of age, and smokers and non-smokers were observed regardless periodontal status (p > 0.05). In contrast, the frequencies of P. aeruginosa and Acinetobacter spp. in saliva and biofilm samples were significantly greater in CP than PH patients (p < 0.01). Smokers presenting P. aeruginosa and high frequencies of supragingival plaque were more likely to present CP than PH. P. aeruginosa and Acinetobacter spp. are frequently detected in the oral microbiota of CP. Poor oral hygiene, smoking and the presence of P. aeruginosa are strongly associated with periodontitis. PMID:25242933

  5. Pseudomonas aeruginosa Biofilm Infections: Community Structure, Antimicrobial Tolerance and Immune Response.

    PubMed

    Rybtke, Morten; Hultqvist, Louise Dahl; Givskov, Michael; Tolker-Nielsen, Tim

    2015-11-20

    Studies of biopsies from infectious sites, explanted tissue and medical devises have provided evidence that biofilms are the underlying cause of a variety of tissue-associated and implant-associated recalcitrant human infections. With a need for novel anti-biofilm treatment strategies, research in biofilm infection microbiology, biofilm formation mechanisms and biofilm-associated antimicrobial tolerance has become an important area in microbiology. Substantial knowledge about biofilm formation mechanisms, biofilm-associated antimicrobial tolerance and immune evasion mechanisms has been obtained through work with biofilms grown in in vitro experimental setups, and the relevance of this information in the context of chronic infections is being investigated by the use of animal models of infection. Because our current in vitro experimental setups and animal models have limitations, new advanced in vitro models developed with knowledge about the chemical landscape at infectious sites are needed. PMID:26319792

  6. Pseudomonas aeruginosa biofilms in disease.

    PubMed

    Mulcahy, Lawrence R; Isabella, Vincent M; Lewis, Kim

    2014-07-01

    Pseudomonas aeruginosa is a ubiquitous organism that is the focus of intense research because of its prominent role in disease. Due to its relatively large genome and flexible metabolic capabilities, this organism exploits numerous environmental niches. It is an opportunistic pathogen that sets upon the human host when the normal immune defenses are disabled. Its deadliness is most apparent in cystic fibrosis patients, but it also is a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disorder, surface growth on implanted biomaterials, and within hospital surface and water supplies, where it poses a host of threats to vulnerable patients (Peleg and Hooper, N Engl J Med 362:1804-1813, 2010; Breathnach et al., J Hosp Infect 82:19-24, 2012). Once established in the patient, P. aeruginosa can be especially difficult to treat. The genome encodes a host of resistance genes, including multidrug efflux pumps (Poole, J Mol Microbiol Biotechnol 3:255-264, 2001) and enzymes conferring resistance to beta-lactam and aminoglycoside antibotics (Vahdani et al., Annal Burns Fire Disast 25:78-81, 2012), making therapy against this gram-negative pathogen particularly challenging due to the lack of novel antimicrobial therapeutics (Lewis, Nature 485: 439-440, 2012). This challenge is compounded by the ability of P. aeruginosa to grow in a biofilm, which may enhance its ability to cause infections by protecting bacteria from host defenses and chemotherapy. Here, we review recent studies of P. aeruginosa biofilms with a focus on how this unique mode of growth contributes to its ability to cause recalcitrant infections. PMID:24096885

  7. Effects of Quorum Sensing Systems on Regulatory T Cells in Catheter-Related Pseudomonas aeruginosa Biofilm Infection Rat Models

    PubMed Central

    Feng, Lei; Xiang, Qingqing; Ai, Qing; Wang, Zhengli; Zhang, Yunhui; Lu, Qi

    2016-01-01

    Background. Quorum sensing (QS) systems play an important role in modulating biofilm formation. Recent studies have found that the QS molecules had complex effects on the host immune systems. In addition, regulatory T cells (Tregs), known as important negative regulators in the immune system, have been found upregulated in multiple chronic infections. Therefore, the QS systems were hypothesized to be involved in modulating Tregs in biofilm-associated infections. Object. To explore the effects of QS systems on Tregs in catheter-related Pseudomonas aeruginosa biofilm infection rat models. Method. Catheter-related Pseudomonas aeruginosa biofilm infection rat models were established; the bacterial clearance rates, total cell counts in bronchoalveolar lavage (BAL) fluid, pathological changes of lungs, and the levels of Foxp3, TGF-β1, and IL-10 in PAO1 strain group were examined and compared with the QS-mutant ΔlasRΔrhlR and ΔlasIΔrhlI groups. Results. In PAO1 group, the bacterial clearance rates were lower, total cell counts were higher, pathological changes were severer, and the levels of Foxp3, TGF-β1, and IL-10 were significantly higher compared with QS-mutant groups (p < 0.05). No significant difference was observed between the two QS-mutant groups (p > 0.05). Conclusion. QS systems can trigger host immune system, accompanied with the upregulation of Tregs. PMID:27069314

  8. Tryptophan Inhibits Biofilm Formation by Pseudomonas aeruginosa

    PubMed Central

    Brandenburg, Kenneth S.; Rodriguez, Karien J.; McAnulty, Jonathan F.; Murphy, Christopher J.; Abbott, Nicholas L.; Schurr, Michael J.

    2013-01-01

    Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the d and l isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of d and l isoforms producing the greatest inhibitory effect. Addition of d-/l-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa. PMID:23318791

  9. Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa

    PubMed Central

    Wei, Qing; Ma, Luyan Z.

    2013-01-01

    Biofilms are communities of microorganisms embedded in extracellular polymeric substances (EPS) matrix. Bacteria in biofilms demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to immune system and antibiotics that render biofilm a source of chronic and persistent infections. A deeper understanding of biofilms will ultimately provide insights into the development of alternative treatment for biofilm infections. The opportunistic pathogen Pseudomonas aeruginosa, a model bacterium for biofilm research, is notorious for its ability to cause chronic infections by its high level of drug resistance involving the formation of biofilms. In this review, we summarize recent advances in biofilm formation, focusing on the biofilm matrix and its regulation in P. aeruginosa, aiming to provide resources for the understanding and control of bacterial biofilms. PMID:24145749

  10. Pseudomonas aeruginosa biofilm: potential therapeutic targets.

    PubMed

    Sharma, Garima; Rao, Saloni; Bansal, Ankiti; Dang, Shweta; Gupta, Sanjay; Gabrani, Reema

    2014-01-01

    Pseudomonas aeruginosa is a gram-negative pathogen that has become an important cause of infection, especially in patients with compromised host defense mechanisms. It is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteremia. The biofilm formed by the bacteria allows it to adhere to any surface, living or non-living and thus Pseudomonal infections can involve any part of the body. Further, the adaptive and genetic changes of the micro-organisms within the biofilm make them resistant to all known antimicrobial agents making the Pseudomonal infections complicated and life threatening. Pel, Psl and Alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell-cell and cell-surface interactions during biofilm formation. Understanding the bacterial virulence which depends on a large number of cell-associated and extracellular factors is essential to know the potential drug targets for future studies. Current novel methods like small molecule based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides, monoclonal antibodies and nanoparticles to curtail the biofilm formed by P. aeruginosa are being discussed in this review. PMID:24309094

  11. Nanoindentation of Pseudomonas aeruginosa bacterial biofilm using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Baniasadi, Mahmoud; Xu, Zhe; Gandee, Leah; Du, Yingjie; Lu, Hongbing; Zimmern, Philippe; Minary-Jolandan, Majid

    2014-12-01

    Bacterial biofilms are a source of many chronic infections. Biofilms and their inherent resistance to antibiotics are attributable to a range of health issues including affecting prosthetic implants, hospital-acquired infections, and wound infection. Mechanical properties of biofilm, in particular, at micro- and nano-scales, are governed by microstructures and porosity of the biofilm, which in turn may contribute to their inherent antibiotic resistance. We utilize atomic force microscopy (AFM)-based nanoindentation and finite element simulation to investigate the nanoscale mechanical properties of Pseudomonas aeruginosa bacterial biofilm. This biofilm was derived from human samples and represents a medically relevant model.

  12. Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Scoffield, Jessica; Silo-Suh, Laura

    2016-08-01

    Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation. PMID:27392247

  13. D-enantiomeric peptides that eradicate wild-type and multi-drug resistant biofilms and protect against lethal Pseudomonas aeruginosa infections

    PubMed Central

    de la Fuente-Núñez, César; Reffuveille, Fany; Mansour, Sarah C.; Reckseidler-Zenteno, Shauna L.; Hernández, Diego; Brackman, Gilles; Coenye, Tom; Hancock, Robert E.W.

    2015-01-01

    SUMMARY In many infections, bacteria form surface-associated communities known as biofilms that are substantially more resistant to antibiotics than their planktonic counterparts. Based on the design features of active anti-biofilm peptides, we made a series of related 12-amino acid L-, D- and retro-inverso derivatives. Specific D-enantiomeric peptides were the most potent at inhibiting biofilm development and eradicating pre-formed biofilms of seven species of wild-type and multiply antibiotic resistant Gram-negative pathogens. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. As shown previously for 1018, these D-amino acid peptides targeted the intracellular stringent response signal (p)ppGpp. The most potent peptides DJK-5 and DJK-6 protected invertebrates from lethal P. aeruginosa infections, and were considerably more active than a previously described L-amino acid peptide 1018. Thus, the protease resistant peptides produced here were more effective both in vitro and in vivo. PMID:25699603

  14. Inhibition of Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa by Culture Extract from Novel Bacterial Species of Paenibacillus Using a Rat Model of Chronic Lung Infection.

    PubMed

    Alasil, Saad Musbah; Omar, Rahmat; Ismail, Salmah; Yusof, Mohd Yasim

    2015-01-01

    Quorum sensing (QS) is a key regulator of virulence factors and biofilm formation in Gram-negative bacteria such as Pseudomonas aeruginosa. Microorganisms that inhabit soil are of strategic importance in the discovery of compounds with anti-QS properties. The objective of the study was to test the culture extract of a taxonomically novel species of Paenibacillus strain 139SI for its inhibitory effects on the QS-controlled virulence factors and biofilm formation of Pseudomonas aeruginosa both in vitro and in vivo. The Paenibacillus sp. culture extract was used to test its anti-QS effects on the LasA protease, LasB elastase, pyoverdin production, and biofilm formation of P. aeruginosa as well as evaluate its therapeutic effects on lung bacteriology, pathology, hematological profile, and serum antibody responses of experimental animals in a rat model of chronic lung infection. Results showed significant decrease in the activities of QS-controlled LasA protease, LasB elastase pyoverdin, and biofilm formation of P. aeruginosa caused by the culture extract. Moreover, the extract significantly prolonged the survival times of rats and facilitated the clearance of biofilm infections from infected lungs. In conclusion, the antiquorum sensing effects of culture extract from a novel species of Paenibacillus provide new insights to combat biofilm-associated infections. PMID:26904749

  15. Inhibition of Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa by Culture Extract from Novel Bacterial Species of Paenibacillus Using a Rat Model of Chronic Lung Infection

    PubMed Central

    Alasil, Saad Musbah; Omar, Rahmat; Yusof, Mohd Yasim

    2015-01-01

    Quorum sensing (QS) is a key regulator of virulence factors and biofilm formation in Gram-negative bacteria such as Pseudomonas aeruginosa. Microorganisms that inhabit soil are of strategic importance in the discovery of compounds with anti-QS properties. The objective of the study was to test the culture extract of a taxonomically novel species of Paenibacillus strain 139SI for its inhibitory effects on the QS-controlled virulence factors and biofilm formation of Pseudomonas aeruginosa both in vitro and in vivo. The Paenibacillus sp. culture extract was used to test its anti-QS effects on the LasA protease, LasB elastase, pyoverdin production, and biofilm formation of P. aeruginosa as well as evaluate its therapeutic effects on lung bacteriology, pathology, hematological profile, and serum antibody responses of experimental animals in a rat model of chronic lung infection. Results showed significant decrease in the activities of QS-controlled LasA protease, LasB elastase pyoverdin, and biofilm formation of P. aeruginosa caused by the culture extract. Moreover, the extract significantly prolonged the survival times of rats and facilitated the clearance of biofilm infections from infected lungs. In conclusion, the antiquorum sensing effects of culture extract from a novel species of Paenibacillus provide new insights to combat biofilm-associated infections. PMID:26904749

  16. Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms.

    PubMed

    Cutruzzolà, Francesca; Frankenberg-Dinkel, Nicole

    2016-01-01

    The formation of the organized bacterial community called biofilm is a crucial event in bacterial physiology. Given that biofilms are often refractory to antibiotics and disinfectants to which planktonic bacteria are susceptible, their formation is also an industrially and medically relevant issue. Pseudomonas aeruginosa, a well-known human pathogen causing acute and chronic infections, is considered a model organism to study biofilms. A large number of environmental cues control biofilm dynamics in bacterial cells. In particular, the dispersal of individual cells from the biofilm requires metabolic and morphological reprogramming in which the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role. The diatomic gas nitric oxide (NO), a well-known signaling molecule in both prokaryotes and eukaryotes, is able to induce the dispersal of P. aeruginosa and other bacterial biofilms by lowering c-di-GMP levels. In this review, we summarize the current knowledge on the molecular mechanisms connecting NO sensing to the activation of c-di-GMP-specific phosphodiesterases in P. aeruginosa, ultimately leading to c-di-GMP decrease and biofilm dispersal. PMID:26260455

  17. Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

    PubMed Central

    2015-01-01

    The formation of the organized bacterial community called biofilm is a crucial event in bacterial physiology. Given that biofilms are often refractory to antibiotics and disinfectants to which planktonic bacteria are susceptible, their formation is also an industrially and medically relevant issue. Pseudomonas aeruginosa, a well-known human pathogen causing acute and chronic infections, is considered a model organism to study biofilms. A large number of environmental cues control biofilm dynamics in bacterial cells. In particular, the dispersal of individual cells from the biofilm requires metabolic and morphological reprogramming in which the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role. The diatomic gas nitric oxide (NO), a well-known signaling molecule in both prokaryotes and eukaryotes, is able to induce the dispersal of P. aeruginosa and other bacterial biofilms by lowering c-di-GMP levels. In this review, we summarize the current knowledge on the molecular mechanisms connecting NO sensing to the activation of c-di-GMP-specific phosphodiesterases in P. aeruginosa, ultimately leading to c-di-GMP decrease and biofilm dispersal. PMID:26260455

  18. Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections.

    PubMed

    Rojo-Molinero, Estrella; Macià, María D; Rubio, Rosa; Moyà, Bartolomé; Cabot, Gabriel; López-Causapé, Carla; Pérez, José L; Cantón, Rafael; Oliver, Antonio

    2016-05-01

    Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t = 2), 4 (t = 4), and 6 days (t = 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one

  19. Differential effects of Pseudomonas aeruginosa on biofilm formation by different strains of Staphylococcus epidermidis.

    PubMed

    Pihl, Maria; Davies, Julia R; Chávez de Paz, Luis E; Svensäter, Gunnel

    2010-08-01

    Pseudomonas aeruginosa and Staphylococcus epidermidis are common opportunistic pathogens associated with medical device-related biofilm infections. 16S rRNA-FISH and confocal laser scanning microscopy were used to study these two bacteria in dual-species biofilms. Two of the four S. epidermidis strains used were shown to form biofilms more avidly on polymer surfaces than the other two strains. In dual-species biofilms, the presence of P. aeruginosa reduced biofilm formation by S. epidermidis, although different clinical isolates differed in their susceptibility to this effect. The most resistant isolate coexisted with P. aeruginosa for up to 18 h and was also resistant to the effects of the culture supernatant from P. aeruginosa biofilms, which caused dispersal from established biofilms of other S. epidermidis strains. Thus, different strains of S. epidermidis differed in their capacity to withstand the action of P. aeruginosa, with some being better equipped than others to coexist in biofilms with P. aeruginosa. Our data suggest that where S. epidermidis and P. aeruginosa are present on abiotic surfaces such as medical devices, S. epidermidis biofilm formation can be inhibited by P. aeruginosa through two mechanisms: disruption by extracellular products, possibly polysaccharides, and, in the later stages, by cell lysis. PMID:20528934

  20. In Vitro Efficacy of a Novel Active-Release Antimicrobial Coating To Eradicate Biofilms of Pseudomonas aeruginosa

    PubMed Central

    Lerdahl, Julia M.; Haymond, Bryan S.; Bloebaum, Roy D.

    2014-01-01

    Implant-related infections are becoming increasingly difficult to treat due to the formation of biofilms on implant surfaces. This study analyzed the in vitro efficacy of a novel antimicrobial coating against biofilms of Pseudomonas aeruginosa, using a flow cell system. Results indicated that P. aeruginosa biofilms were reduced by greater than 8 log10 units in less than 24 h. Data indicated that this active-release coating may be promising for preventing biofilm implant-related infections. PMID:24395238

  1. Comparison of UVB and UVC irradiation disinfection efficacies on Pseudomonas Aeruginosa (P. aeruginosa) biofilm

    NASA Astrophysics Data System (ADS)

    Argyraki, A.; Markvart, M.; Nielsen, Anne; Bjarnsholt, T.; Bjørndal, L.; Petersen, P. M.

    2016-04-01

    Disinfection routines are important in all clinical applications. The uprising problem of antibiotic resistance has driven major research efforts towards alternative disinfection approaches, involving light-based solutions. Pseudomonas aeruginosa (P. aeruginosa) is a common bacterium that can cause skin, soft tissue, lungs, kidney and urinary tract infections. Moreover, it can be found on and in medical equipment causing often cross infections in hospitals. The objective of this study was to test the efficiency, of two different light-based disinfection treatments, namely UVB and UVC irradiation, on P. aeruginosa biofilms at different growth stages. In our experiments a new type of UV light emitting diodes (LEDs) were used to deliver UV irradiation on the biofilms, in the UVB (296nm) and UVC (266nm) region. The killing rate was studied as a function of dose for 24h grown biofilms. The dose was ramped from 72J/m2 to 10000J/m2. It was shown that UVB irradiation was more effective than UVC irradiation in inactivating P. aeruginosa biofilms. No colony forming units (CFU) were observed for the UVB treated biofilms when the dose was 10000 J/m2 (CFU in control sample: 7.5 x 104). UVB irradiation at a dose of 20000J/m2 on mature biofilms (72h grown) resulted in a 3.9 log killing efficacy. The fact that the wavelength of 296nm exists in daylight and has such disinfection ability on biofilms gives new perspectives for applications within disinfection at hospitals.

  2. Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets.

    PubMed

    Hassett, Daniel J; Cuppoletti, John; Trapnell, Bruce; Lymar, Sergei V; Rowe, John J; Yoon, Sang Sun; Hilliard, George M; Parvatiyar, Kislay; Kamani, Moneesha C; Wozniak, Daniel J; Hwang, Sung Hei; McDermott, Timothy R; Ochsner, Urs A

    2002-12-01

    Recent evidence indicates that Pseudomonas aeruginosa residing as biofilms in airway mucus of cystic fibrosis (CF) patients is undergoing anaerobic metabolism, a form of growth requiring gene products that are not utilized during aerobic growth. The outer membrane protein, OprF, and the rhl quorum sensing circuit are two previously unrecognized cellular factors that are required for optimal anaerobic biofilm viability. Without OprF, bacteria grow extremely poorly because they lack nitrite reductase activity while lacking rhlR or rhlI forces bacteria to undergo metabolic suicide by overproduction of nitric oxide. Furthermore, anaerobic growth favors maintenance of the mucoid, alginate-overproducing phenotype. Thus, with increasing age of CF patients, mucoid populations predominate, indicating that anaerobic bacteria reside in the inspissated airway mucus. Because many frontline antibiotics used in the treatment of CF airway disease are either ineffective or show reduced efficacy during anaerobic conditions, we propose development of new drugs to combat anaerobic metabolism by P. aeruginosa for more effective treatment of chronic CF lung infections. PMID:12458153

  3. Biophysics of Biofilm Infection

    PubMed Central

    Stewart, Philip S.

    2014-01-01

    This article examines a likely basis of the tenacity of biofilm infections that has received relatively little attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength exceeds the external applied stress. Mechanical failure of the biofilm and release of planktonic microbial cells is also important in vivo because it can result in dissemination of infection. The fundamental criterion for detachment and dissemination is that the applied stress exceeds the biofilm failure strength. The apparent contradiction for a biofilm to both persist and disseminate is resolved by recognizing that biofilm material properties are inherently heterogeneous. There are also mechanical aspects to the ways that infectious biofilms evade leukocyte phagocytosis. The possibility of alternative therapies for treating biofilm infections that work by reducing biofilm cohesion could: 1) allow prevailing hydrodynamic shear to remove biofilm, 2) increase the efficacy of designed interventions for removing biofilms, 3) enable phagocytic engulfment of softened biofilm aggregates, and 4) improve phagocyte mobility and access to biofilm. PMID:24376149

  4. Biophysics of biofilm infection.

    PubMed

    Stewart, Philip S

    2014-04-01

    This article examines a likely basis of the tenacity of biofilm infections that has received relatively little attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength exceeds the external applied stress. Mechanical failure of the biofilm and release of planktonic microbial cells is also important in vivo because it can result in dissemination of infection. The fundamental criterion for detachment and dissemination is that the applied stress exceeds the biofilm failure strength. The apparent contradiction for a biofilm to both persist and disseminate is resolved by recognizing that biofilm material properties are inherently heterogeneous. There are also mechanical aspects to the ways that infectious biofilms evade leukocyte phagocytosis. The possibility of alternative therapies for treating biofilm infections that work by reducing biofilm cohesion could (1) allow prevailing hydrodynamic shear to remove biofilm, (2) increase the efficacy of designed interventions for removing biofilms, (3) enable phagocytic engulfment of softened biofilm aggregates, and (4) improve phagocyte mobility and access to biofilm. PMID:24376149

  5. Mechanical destruction of pseudomonas aeruginosa biofilms by ultrasound exposure

    NASA Astrophysics Data System (ADS)

    Xu, Jin; Bigelow, Timothy A.; Halverson, Larry J.; Middendorf, Jill; Rusk, Ben

    2012-10-01

    Medical implants are prone to colonization by bacterial biofilms, which are highly resistant to antibiotics. Normally, surgery is required to replace the infected implant. One promising non-invasive treatment option is to destroy the biofilm with high-intensity focused ultrasound (HIFU) exposure. In our study, Pseudomonas aeruginosa bacterial biofilms were grown on graphite disks in a flow chamber for three days prior to exposing them to ultrasound pulses of varying duration or burst period. The pulses were 20 cycles in duration at a frequency of 1.1 MHz from a spherically focused transducer (f/1, 63 mm focal length), creating peak compressional and rarefactional pressures at the disk surface of 30 and 13 MPa, respectively. P. aeruginosa were tagged with GFP and cells killed by HIFU were visualized using propidium iodide, which permeates membranes of dead cells, to aid determining the extent of biofilm destruction and whether cells are alive or dead. Our results indicate that a 30-s exposure and 6-ms pulse period or those combinations with the same number of pulses, were sufficient to destroy the biofilm and to kill the remaining cells. Reducing the number of pulses decreased biofilm destruction, leaving more dead and live bacteria on the surface.

  6. Spaceflight promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Kim, Wooseong; Tengra, Farah K; Young, Zachary; Shong, Jasmine; Marchand, Nicholas; Chan, Hon Kit; Pangule, Ravindra C; Parra, Macarena; Dordick, Jonathan S; Plawsky, Joel L; Collins, Cynthia H

    2013-01-01

    Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight. PMID:23658630

  7. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    PubMed Central

    Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014

  8. Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1.

    PubMed

    Nithya, Chari; Begum, Mansur Farzana; Pandian, Shunmugiah Karutha

    2010-09-01

    According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 microg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70-74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat. PMID:20665017

  9. Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa.

    PubMed

    Yang, Liang; Liu, Yang; Markussen, Trine; Høiby, Niels; Tolker-Nielsen, Tim; Molin, Søren

    2011-08-01

    Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very important for understanding of biofilm physiology and the treatment of biofilm-related infectious diseases. Here, we have investigated interactions of two of the major bacterial species of cystic fibrosis lung microbial communities -Pseudomonas aeruginosa and Staphylococcus aureus- when grown in co-culture biofilms. By growing co-culture biofilms of S. aureus with P. aeruginosa mutants in a flow-chamber system and observing them using confocal laser scanning microscopy, we show that wild-type P. aeruginosa PAO1 facilitates S. aureus microcolony formation. In contrast, P. aeruginosa mucA and rpoN mutants do not facilitate S. aureus microcolony formation and tend to outcompete S. aureus in co-culture biofilms. Further investigations reveal that extracellular DNA (eDNA) plays an important role in S. aureus microcolony formation and that P. aeruginosa type IV pili are required for this process, probably through their ability to bind to eDNA. Furthermore, P. aeruginosa is able to protect S. aureus against Dictyostelium discoideum phagocytosis in co-culture biofilms. PMID:21595754

  10. A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions.

    PubMed

    Alves, Diana R; Perez-Esteban, P; Kot, W; Bean, J E; Arnot, T; Hansen, L H; Enright, Mark C; Jenkins, A Tobias A

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities - biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred. PMID:26347362

  11. Cooperative pathogenicity in cystic fibrosis: Stenotrophomonas maltophilia modulates Pseudomonas aeruginosa virulence in mixed biofilm

    PubMed Central

    Pompilio, Arianna; Crocetta, Valentina; De Nicola, Serena; Verginelli, Fabio; Fiscarelli, Ersilia; Di Bonaventura, Giovanni

    2015-01-01

    The present study was undertaken in order to understand more about the interaction occurring between S. maltophilia and P. aeruginosa, which are frequently co-isolated from CF airways. For this purpose, S. maltophilia RR7 and P. aeruginosa RR8 strains, co-isolated from the lung of a chronically infected CF patient during a pulmonary exacerbation episode, were evaluated for reciprocal effect during planktonic growth, adhesion and biofilm formation onto both polystyrene and CF bronchial cell monolayer, motility, as well as for gene expression in mixed biofilms. P. aeruginosa significantly affected S. maltophilia growth in both planktonic and biofilm cultures, due to an inhibitory activity probably requiring direct contact. Conversely, no effect was observed on P. aeruginosa by S. maltophilia. Compared with monocultures, the adhesiveness of P. aeruginosa on CFBE41o- cells was significantly reduced by S. maltophilia, which probably acts by reducing P. aeruginosa's swimming motility. An opposite trend was observed for biofilm formation, confirming the findings obtained using polystyrene. When grown in mixed biofilm with S. maltophilia, P. aeruginosa significantly over-expressed aprA, and algD—codifying for protease and alginate, respectively—while the quorum sensing related rhlR and lasI genes were down-regulated. The induced alginate expression by P. aeruginosa might be responsible for the protection of S. maltophilia against tobramycin activity we observed in mixed biofilms. Taken together, our results suggest that the existence of reciprocal interference of S. maltophilia and P. aeruginosa in CF lung is plausible. In particular, S. maltophilia might confer some selective “fitness advantage” to P. aeruginosa under the specific conditions of chronic infection or, alternatively, increase the virulence of P. aeruginosa thus leading to pulmonary exacerbation. PMID:26441885

  12. In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.

    PubMed

    Fernández-Olmos, Ana; García-Castillo, María; Maiz, Luis; Lamas, Adelaida; Baquero, Fernando; Cantón, Rafael

    2012-08-01

    The ability of antibiotics used in bronchopulmonary infections in cystic fibrosis (CF) patients to prevent Pseudomonas aeruginosa early biofilm formation was studied using a biofilm microtitre assay with 57 non-mucoid P. aeruginosa isolates (44 first colonisers and 13 recovered during the initial intermittent colonisation stage) obtained from 35 CF patients. Minimum biofilm inhibitory concentrations (BICs) of levofloxacin, ciprofloxacin, imipenem, ceftazidime, tobramycin, colistin and azithromycin were determined by placing a peg lid with a formed biofilm onto microplates containing antibiotics. A modification of this protocol consisting of antibiotic challenge during biofilm formation was implemented in order to determine the biofilm prevention concentration (BPC), i.e. the minimum concentration able to prevent biofilm formation. The lowest BPCs were for fluoroquinolones, tobramycin and colistin and the highest for ceftazidime and imipenem. The former antibiotics had BPCs identical to or only slightly higher than their minimum inhibitory concentrations (MICs) determined by standard Clinical and Laboratory Standards Institute (CLSI) microdilution and were also active on formed biofilms as reflected by their low BIC values. In contrast, ceftazidime and imipenem were less effective for prevention of biofilm formation and on formed biofilms. In conclusion, the new BPC parameter determined in non-mucoid P. aeruginosa isolates recovered during early colonisation stages in CF patients supports early aggressive antimicrobial treatment guidelines in first P. aeruginosa-colonised CF patients. PMID:22727530

  13. Novel Inhaled Combination Powder Containing Amorphous Colistin and Crystalline Rifapentine with Enhanced Antimicrobial Activities against Planktonic Cells and Biofilm of Pseudomonas aeruginosa for Respiratory Infections.

    PubMed

    Zhou, Qi Tony; Sun, Si-Ping; Chan, John Gar Yan; Wang, Ping; Barraud, Nicolas; Rice, Scott A; Wang, Jiping; Li, Jian; Chan, Hak-Kim

    2015-08-01

    Colistin has been increasingly used for the treatment of respiratory infections caused by Gram-negative bacteria. Unfortunately parenteral administration of colistin can cause severe adverse effects. This study aimed to develop an inhaled combination dry powder formulation of colistin and rifapentine for the treatment of respiratory infections. The combination formulation was produced by spray-drying rifapentine particles suspended in an aqueous colistin solution. The combination dry powder had enhanced antimicrobial activities against planktonic cells and biofilm cultures of Pseudomonas aeruginosa, with both minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values (2 and 4 mg/L, respectively) being half that of pure colistin (MIC 4 mg/L and MBIC 8 mg/L) and 1/16th that of pure rifapentine (MIC 32 mg/L and MBIC 64 mg/L). High aerosol performance, as measured via an Aerolizer device, was observed with emitted doses>89% and fine particle fraction (FPF) total>76%. The proportion of submicron particles of rifapentine particles was minimized by the attachment of colistin, which increased the overall particle mass and aerodynamic size distribution. Using the spray-drying method described here, stable particles of amorphous colistin and crystalline rifapentine were distributed homogeneously in each stage of the impinger. Unlike the colistin alone formulation, no deterioration in aerosol performance was found for the combination powder when exposed to a high relative humidity of 75%. In our previous study, surface coating by rifampicin contributed to the moisture protection of colistin. Here, a novel approach with a new mechanism was proposed whereby moisture protection was attributed to the carrier effect of elongated crystalline rifapentine particles, which minimized contact between hygroscopic colistin particles. This inhaled combination antibiotic formulation with enhanced aerosol dispersion efficiency and in vitro efficacy

  14. The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms

    PubMed Central

    2015-01-01

    P. aeruginosa is an opportunistic pathogenic bacterium responsible for both acute and chronic infections. Beyond its natural resistance to many drugs, its ability to form biofilm, a complex biological system, renders ineffective the clearance by immune defense systems and antibiotherapy. The objective of this report is to provide an overview (i) on P. aeruginosa biofilm lifestyle cycle, (ii) on the main key actors relevant in the regulation of biofilm formation by P. aeruginosa including QS systems, GacS/GacA and RetS/LadS two-component systems and C-di-GMP-dependent polysaccharides biosynthesis, and (iii) finally on reported natural and synthetic products that interfere with control mechanisms of biofilm formation by P. aeruginosa without affecting directly bacterial viability. Concluding remarks focus on perspectives to consider biofilm lifestyle as a target for eradication of resistant infections caused by P. aeruginosa. PMID:25866808

  15. Biofilm Formation Mechanisms of Pseudomonas aeruginosa Predicted via Genome-Scale Kinetic Models of Bacterial Metabolism

    PubMed Central

    Vital-Lopez, Francisco G.; Reifman, Jaques; Wallqvist, Anders

    2015-01-01

    A hallmark of Pseudomonas aeruginosa is its ability to establish biofilm-based infections that are difficult to eradicate. Biofilms are less susceptible to host inflammatory and immune responses and have higher antibiotic tolerance than free-living planktonic cells. Developing treatments against biofilms requires an understanding of bacterial biofilm-specific physiological traits. Research efforts have started to elucidate the intricate mechanisms underlying biofilm development. However, many aspects of these mechanisms are still poorly understood. Here, we addressed questions regarding biofilm metabolism using a genome-scale kinetic model of the P. aeruginosa metabolic network and gene expression profiles. Specifically, we computed metabolite concentration differences between known mutants with altered biofilm formation and the wild-type strain to predict drug targets against P. aeruginosa biofilms. We also simulated the altered metabolism driven by gene expression changes between biofilm and stationary growth-phase planktonic cultures. Our analysis suggests that the synthesis of important biofilm-related molecules, such as the quorum-sensing molecule Pseudomonas quinolone signal and the exopolysaccharide Psl, is regulated not only through the expression of genes in their own synthesis pathway, but also through the biofilm-specific expression of genes in pathways competing for precursors to these molecules. Finally, we investigated why mutants defective in anthranilate degradation have an impaired ability to form biofilms. Alternative to a previous hypothesis that this biofilm reduction is caused by a decrease in energy production, we proposed that the dysregulation of the synthesis of secondary metabolites derived from anthranilate and chorismate is what impaired the biofilms of these mutants. Notably, these insights generated through our kinetic model-based approach are not accessible from previous constraint-based model analyses of P. aeruginosa biofilm

  16. The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance of Pseudomonas aeruginosa.

    PubMed

    Oglesby-Sherrouse, Amanda G; Djapgne, Louise; Nguyen, Angela T; Vasil, Adriana I; Vasil, Michael L

    2014-04-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment for such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here, we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, nonsiderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by subinhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development. PMID:24436170

  17. Effects of clinical isolates of Pseudomonas aeruginosa on Staphylococcus epidermidis biofilm formation.

    PubMed

    Pihl, Maria; Chávez de Paz, Luis E; Schmidtchen, Artur; Svensäter, Gunnel; Davies, Julia R

    2010-08-01

    Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonization by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA FISH and confocal laser scanning microscopy. Among the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect, and even after 6 h, S. epidermidis levels in dual-species biofilms were reduced by >85% compared with those without P. aeruginosa. Interestingly, strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa, which may develop over time in chronic infections, could counteract colonization by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon. PMID:20579097

  18. In Vivo Efficacy of Antimicrobials against Biofilm-Producing Pseudomonas aeruginosa.

    PubMed

    Pawar, Vinay; Komor, Uliana; Kasnitz, Nadine; Bielecki, Piotr; Pils, Marina C; Gocht, Benjamin; Moter, Annette; Rohde, Manfred; Weiss, Siegfried; Häussler, Susanne

    2015-08-01

    Patients suffering from cystic fibrosis (CF) are commonly affected by chronic Pseudomonas aeruginosa biofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate that P. aeruginosa is able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling of P. aeruginosa indicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment of P. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-type P. aeruginosa PA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number of P. aeruginosa PA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds. PMID:26055372

  19. The effects of D-Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa.

    PubMed

    She, Pengfei; Chen, Lihua; Liu, Hongbo; Zou, Yaru; Luo, Zhen; Koronfel, Asmaa; Wu, Yong

    2015-09-01

    The biofilm formation of microorganisms causes persistent tissue infections resistant to treatment with antimicrobial agents. Pseudomonas aeruginosa is commonly isolated from the airways of patients with chronic fibrosis (CF) and often forms biofilms, which are extremely hard to eradicate and a major cause of mortality and morbidity. Recent studies have shown that D-amino acids (D-AAs) inhibited and disrupted biofilm formation by causing the release of the protein component of the polymeric matrix. However, the effects of D-AAs combined with common antibiotics on biofilms have rarely been studied. The current study first determined whether D-AAs disrupted the biofilms of PAO1 and the clinical airway isolates of P. aeruginosa. It was then determined whether combinations of D-Tyr (the most effective one) and the antibiotic amikacin (AMK) enhanced the activity against these biofilms. The results of the current study showed that D-Tyr is the most effective among those that disassemble the D-amino acids (D-leucine, D-methionine, D-Tyrptophan, and D-tryptophan), and D-Tyr at concentrations higher than 5 mM significantly reduced the biofilm biomass of P. aeruginosa (p < 0.05) without influencing bacterial growth. It was also revealed that D-Tyr improved the efficacy of AMK to combat P. aeruginosa biofilms, as indicated by a reduction in the minimal biofilm-inhibiting concentration (MBIC50 and MBIC90) without a change in the minimal inhibitory concentration (MIC) of planktonic bacteria. Thus, the findings indicated that D-Tyr supplementation overcame the resistance of P. aeruginosa biofilms to AMK, which might be helpful for preventing AMK overuse when this specific D-Tyr is recommended for combatting these biofilms. Also, toxicity of the liver and kidney from AMK could be potentially mitigated by co-delivery with D-Tyr. PMID:26188263

  20. Sodium Nitrite Blocks the Activity of Aminoglycosides against Pseudomonas aeruginosa Biofilms

    PubMed Central

    Zemke, Anna C.; Gladwin, Mark T.

    2015-01-01

    Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 μg/ml to >1,024 μg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm. PMID:25801569

  1. [Structural components and peculiarities of Pseudomonas aeruginosa biofilm organization].

    PubMed

    Balko, O B; Avdieieva, L V

    2010-01-01

    Peculiarities of the structural organization of bacterial biofilm during its formation and disintegration have been investigated on the model of Pseudomonas aeruginosa UCM B-900 (ATCC 9027). It was shown, that development of the biofilm in a stationary system on glass was a two-vector process with changes in time and space. P. aeruginosa UCM B-900 biofilm is formed from single cells, passes through the stages of base components, net structure, islands and comes to the end with integration into a complete monolayer. The biofilm degradation repeats the stages of its formation in the reverse sequence. PMID:20812507

  2. Inhibition of Pseudomonas aeruginosa biofilm formation by 2,2’-bipyridyl, lipoic, kojic and picolinic acids

    PubMed Central

    Çevik, Kübra; Ulusoy, Seyhan

    2015-01-01

    Objective(s): The inhibitory effects of iron chelators, and FeCl3 chelation on biofilm formation and swarming motility were investigated against an opportunistic human pathogen Pseudomonas aeruginosa. Materials and Methods: The inhibitory activity of 2,2’-bipyridyl, lipoic acid, kojic acid and picolinic acid on biofilm formation of P. aeruginosa strain PAO1 and three clinical isolates (P. aeruginosa PAK01, P. aeruginosa PAK02 and P. aeruginosa PAK03) were investigated, based on crystal violet assay, and swarming motility test. Results: The kojic, lipoic and picolinic acid inhibited biofilm formation by 5-33% in all tested P. aeruginosa isolates. When chelated iron was added, biofilm inhibition rates were determined to be 39-57%. Among the tested chelators against P. aeruginosa, lipoic acid (84%) and kojic acid (68%) presented the highest inhibition of swarming motility. This is the first study to report the inhibitory effect of lipoic acid on biofilm formation and swarming motility of P. aeruginosa. Conclusion: It is considered that lipoic and picolinic acids can serve as alternatives for the treatment of the P. aeruginosa infections by inhibiting biofilm formation. PMID:26557964

  3. Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Hammond, John H.; Dolben, Emily F.; Smith, T. Jarrod; Bhuju, Sabin

    2015-01-01

    ABSTRACT In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections. IMPORTANCE Pseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently

  4. Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.

    PubMed

    Kolpen, Mette; Mousavi, Nabi; Sams, Thomas; Bjarnsholt, Thomas; Ciofu, Oana; Moser, Claus; Kühl, Michael; Høiby, Niels; Jensen, Peter Østrup

    2016-02-01

    Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterised by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leucocyte activity. Whilst the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficacy of several bactericidal antibiotics such as ciprofloxacin is enhanced by stimulation of the aerobic respiration of pathogens, and that lack of O2 increases their tolerance. Reoxygenation of O2-depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. We tested such a strategy using reoxygenation of O2-depleted P. aeruginosa strain PAO1 agarose-embedded biofilms by hyperbaric oxygen treatment (HBOT) (100% O2, 2.8bar), enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. This proof-of-principle study demonstrates that biofilm reoxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections. PMID:26774522

  5. Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions.

    PubMed

    Kolpen, Mette; Appeldorff, Cecilie F; Brandt, Sarah; Mousavi, Nabi; Kragh, Kasper N; Aydogan, Sevtap; Uppal, Haleema A; Bjarnsholt, Thomas; Ciofu, Oana; Høiby, Niels; Jensen, Peter Ø

    2016-02-01

    Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH ˙ may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions. PMID:26458402

  6. Pseudomonas aeruginosa Cystic Fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro

    PubMed Central

    2010-01-01

    Background Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. Results Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. Conclusions Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung. PMID:20141637

  7. Inhibition of Biofilm Formation by Esomeprazole in Pseudomonas aeruginosa and Staphylococcus aureus

    PubMed Central

    Singh, Vandana; Arora, Vaneet; Alam, M. Jahangir

    2012-01-01

    Staphylococcus aureus and Pseudomonas aeruginosa are common nosocomial pathogens responsible for biofilm-associated infections. Proton pump inhibitors (PPI), such as esomeprazole, may have novel antimicrobial properties. The objective of this study was to assess whether esomeprazole prevents sessile bacterial growth and biofilm formation and whether it may have synergistic killing effects with standard antibiotics. The antibiofilm activity of esomeprazole at 0.25 mM was tested against two strains each of S. aureus and P. aeruginosa. Bacterial biofilms were prepared using a commercially available 96-peg-plate Calgary biofilm device. Sessile bacterial CFU counts and biomass were assessed during 72 hours of esomeprazole exposure. The killing activities after an additional 24 hours of vancomycin (against S. aureus) and meropenem (against P. aeruginosa) treatment with or without preexposure to esomeprazole were also assessed by CFU and biomass analyses. P. aeruginosa and S. aureus strains exposed to esomeprazole displayed decreased sessile bacterial growth and biomass (P < 0.001, each parameter). After 72 h of exposure, there was a 1-log10 decrease in the CFU/ml of esomeprazole-exposed P. aeruginosa and S. aureus strains compared to controls (P < 0.001). After 72 h of exposure, measured absorbance was 100% greater in P. aeruginosa control strains than in esomeprazole-exposed strains (P < 0.001). Increased killing and decreased biomass were observed for esomeprazole-treated bacteria compared to untreated controls exposed to conventional antibiotics (P < 0.001, each parameter). Reduced biofilm growth after 24 h was visibly apparent by light micrographs for P. aeruginosa and S. aureus isolates exposed to esomeprazole compared to untreated controls. In conclusion, esomeprazole demonstrated an antibiofilm effect against biofilm-producing S. aureus and P. aeruginosa. PMID:22664967

  8. Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation.

    PubMed

    Price, Katherine E; Orazi, Giulia; Ruoff, Kathryn L; Hebert, Wesley P; O'Toole, George A; Mastoridis, Paul

    2015-01-01

    Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting. PMID:26506004

  9. Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

    PubMed Central

    Sauer, Karin; Camper, Anne K.; Ehrlich, Garth D.; Costerton, J. William; Davies, David G.

    2002-01-01

    Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth. PMID:11807075

  10. Incorporation of Farnesol Significantly Increases the Efficacy of Liposomal Ciprofloxacin against Pseudomonas aeruginosa Biofilms in Vitro.

    PubMed

    Bandara, H M H N; Herpin, M J; Kolacny, D; Harb, A; Romanovicz, D; Smyth, H D C

    2016-08-01

    The challenge of eliminating Pseudomonas aeruginosa infections, such as in cystic fibrosis lungs, remains unchanged due to the rapid development of antibiotic resistance. Poor drug penetration into dense P. aeruginosa biofilms plays a vital role in ineffective clearance of the infection. Thus, the current antibiotic therapy against P. aeruginosa biofilms need to be revisited and alternative antibiofilm strategies need to be invented. Fungal quorum sensing molecule (QSM), farnesol, appears to have detrimental effects on P. aeruginosa. Thus, this study aimed to codeliver naturally occurring QSM farnesol, with the antibiotic ciprofloxacin as a liposomal formulation to eradicate P. aeruginosa biofilms. Four different liposomes (with ciprofloxacin and farnesol, Lcip+far; with ciprofloxacin, Lcip; with farnesol, Lfar; control, Lcon) were prepared using dehydration-rehydration method and characterized. Drug entrapment and release were evaluated by spectrometry and high performance liquid chromatography (HPLC). The efficacy of liposomes was assessed using standard biofilm assay. Liposome-treated 24 h P. aeruginosa biofilms were quantitatively assessed by XTT reduction assay and crystal violet assay, and qualitatively by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Ciprofloxacin release from liposomes was higher when encapsulated with farnesol (Lcip+far) compared to Lcip (3.06% vs 1.48%), whereas farnesol release was lower when encapsulated with ciprofloxacin (Lcip+far) compared to Lfar (1.81% vs 4.75%). The biofilm metabolism was significantly lower when treated with Lcip+far or Lcip compared to free ciprofloxacin (XTT, P < 0.05). When administered as Lcip+far, the ciprofloxacin concentration required to achieve similar biofilm inhibition was 125-fold or 10-fold lower compared to free ciprofloxacin or Lcip, respectively (P < 0.05). CLSM and TEM confirmed predominant biofilm disruption, greater dead cell ratio, and increased depth of

  11. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

    PubMed Central

    Wilton, Mike; Charron-Mazenod, Laetitia; Moore, Richard

    2015-01-01

    Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid l-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype. PMID:26552982

  12. [Urinary catheter biofilm infections].

    PubMed

    Holá, V; Růzicka, F

    2008-04-01

    Urinary tract infections, most of which are biofilm infections in catheterized patients, account for more than 40% of hospital infections. Bacterial colonization of the urinary tract and catheters causes not only infection but also other complications such as catheter blockage by bacterial encrustation, urolithiasis and pyelonephritis. About 50% of long-term catheterized patients face urinary flow obstruction due to catheter encrustation, but no measure is currently available to prevent it. Encrustation has been known either to result from metabolic dysfunction or to be of microbial origin, with urease positive bacterial species implicated most often. Infectious calculi account for about 15-20% of all cases of urolithiasis and are often associated with biofilm colonization of a long-term indwelling urinary catheter or urethral stent. The use of closed catheter systems is helpful in reducing such problems; nevertheless, such a system only delays the inevitable, with infections emerging a little later. Various coatings intended to prevent the bacterial adhesion to the surface of catheters and implants and thus also the emergence of biofilm infections, unfortunately, do not inhibit the microbial adhesion completely and permanently and the only reliable method for biofilm eradication remains the removal of the foreign body from the patient. PMID:18578409

  13. Inhibition of Pseudomonas aeruginosa biofilm formation on wound dressings

    PubMed Central

    Brandenburg, Kenneth S.; Calderon, Diego F.; Kierski, Patricia R.; Brown, Amanda L.; Shah, Nihar M.; Abbott, Nicholas L.; Schurr, Michael J.; Murphy, Christopher J.; McAnulty, Jonathan F.; Czuprynski, Charles J.

    2016-01-01

    Chronic non-healing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building upon prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the 3-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing. PMID:26342168

  14. Ambroxol inhibits mucoid conversion of Pseudomonas aeruginosa and contributes to the bactericidal activity of ciprofloxacin against mucoid P. aeruginosa biofilms.

    PubMed

    Wang, Wenlei; Yu, Jialin; He, Yu; Wang, Zhengli; Li, Fang

    2016-07-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that can cause severe infections in immunocompromised individuals. Because it forms biofilms, which protect against host immune attack and increase resistance to conventional antibiotics, mucoid P. aeruginosa is nearly impossible to eradicate. Moreover, mucoid conversion of P. aeruginosa in cystic fibrosis (CF) patients leads to poor outcomes. This conversion is mainly due to mucA gene mutation, which is thought to be induced by polymorphonuclear leukocytes (PMNs) and the reactive oxygen species they release. Ambroxol, a mucolytic agent with antioxidant characteristics, is used clinically, and this compound has recently been demonstrated to possess anti-biofilm properties. In this study, we found that ambroxol inhibits the H2 O2 -mediated conversion of P. aeruginosa from a non-mucoid to a mucoid phenotype, an effect that is due to its antioxidant property against H2 O2 . Furthermore, the bactericidal activity of ciprofloxacin against mucoid P. aeruginosa biofilms was increased in vitro when used in combination with ambroxol. PMID:27102839

  15. Potential of Ocimum basilicum L. and Salvia officinalis L. essential oils against biofilms of P. aeruginosa clinical isolates.

    PubMed

    Stojanović-Radić, Z; Pejcić, M; Stojanović, N; Sharifi-Rad, J; Stanković, N

    2016-01-01

    Biofilms are complex communities of microorganisms, responsible for more than 60% of the chronic human infections and they represent one of the leading concerns in medicine. Pseudomonas aeruginosa is human pathogenic bacteria which causes numerous diseases and is known for its ability to produce biofilm. Ocimum basilicum L. (basil) and Salvia officinalis L. (sage) are widely used plants in traditional medicine for the treatment of different conditions. Therefore, the aim of this study was to investigate the potential of basil and sage essential oils against P. aeruginosa biofilm producing strains. The efficacy of two essential oils on P. aeruginosa biofilm forming ability was determined using crystal violet method. Out of 15 strains isolated from different clinical biological samples, two were strong, 11 moderate and one weak biofilm producer. Good efficacy of sage essential oil towards strong and weak biofilm producers, but not of basil essential oil, was observed. In the case of moderate biofilm producers, 81.8% showed lower biofilm production after incubation with the sage oil, while 63.6% showed the reduction of biofilm production after basil essential oil treatment. The obtained results showed high potential of both oils for the treatment of persistent infections caused by Pseudomonas aeruginosa biofilms. PMID:27585258

  16. Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

    PubMed Central

    Das, Theerthankar; Kutty, Samuel K.; Tavallaie, Roya; Ibugo, Amaye I.; Panchompoo, Janjira; Sehar, Shama; Aldous, Leigh; Yeung, Amanda W. S.; Thomas, Shane R.; Kumar, Naresh; Gooding, J. Justin; Manefield, Mike

    2015-01-01

    Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation. PMID:25669133

  17. In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states.

    PubMed

    Velez Perez, Antonio L; Schmidt-Malan, Suzannah M; Kohner, Peggy C; Karau, Melissa J; Greenwood-Quaintance, Kerryl E; Patel, Robin

    2016-07-01

    Pseudomonas aeruginosa causes a variety of life-threatening infections, some of which are associated with planktonic and others with biofilm states. Herein, we tested the combination of the novel cephalosporin, ceftolozane, with the β-lactamase inhibitor, tazobactam, against planktonic and biofilm forms of 54 clinical isolates of P. aeruginosa, using cefepime as a comparator. MIC values were determined following Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum biofilm inhibitory concentration (MBIC) values were determined using biofilm-laden pegged lids incubated in antimicrobial challenge plates containing varying concentrations of ceftolozane/tazobactam. Pegged lids were then incubated in growth recovery plates containing cation-adjusted Mueller-Hinton broth to determine the minimum biofilm bactericidal concentration (MBBC). Ceftolozane/tazobactam was highly active against planktonic P. aeruginosa, with all 54 isolates studied testing susceptible (MIC ≤4/4μg/mL). On the other hand, 51/54 biofilm P. aeruginosa had MBICs ≥16/4μg/mL, and all 54 isolates had MBBCs >32μg/mL. Of the 54 isolates, 45 (83.3%) tested susceptible to cefepime, with the MIC50/MIC90 being 4/16μg/mL, respectively, and the MBIC90 and MBBC90 both being >256μg/mL. Although ceftolozane/tazobactam is a promising antimicrobial agent for the treatment of P. aeruginosa infections, it is not highly active against P. aeruginosa biofilms. PMID:27130477

  18. Chemotaxis in P. Aeruginosa Biofilm Formation

    NASA Astrophysics Data System (ADS)

    Bienvenu, Samuel; Strain, Shinji; Thatcher, Travis; Gordon, Vernita

    2010-10-01

    Pseudomonas biofilms form infections in the lungs of Cystic Fibrosis (CF) patients that damage lung tissue and lead to death. Previous work shows chemotaxis is important for Pseudomonas in CF lungs. The work studied swimming bacteria at high concentrations. In contrast, medically relevant biofilms initiate from sparse populations of surface-bound bacteria. The recent development of software techniques for automated, high-throughput bacteria tracking leaves us well-poised to quantitatively study these chemotactic conditions. We will develop experimental systems for such studies, focusing on L-Arginine (an amino acid), D-Galactose (a sugar present in lungs), and succinate and glucose (carbon sources for bacteria). This suite of chemoattractants will allow us to study how chemoattractant characteristics--size and diffusion behavior--change bacterial response; the interaction of competing chemoattractants; and, differences in bacterial behaviors, like motility modes, in response to different types of chemoattractions and varying neighbor cell density.

  19. Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

    PubMed Central

    Trejo-Hernández, Abigail; Andrade-Domínguez, Andrés; Hernández, Magdalena; Encarnación, Sergio

    2014-01-01

    Inter-kingdom and interspecies interactions are ubiquitous in nature and are important for the survival of species and ecological balance. The investigation of microbe-microbe interactions is essential for understanding the in vivo activities of commensal and pathogenic microorganisms. Candida albicans, a polymorphic fungus, and Pseudomonas aeruginosa, a Gram-negative bacterium, are two opportunistic pathogens that interact in various polymicrobial infections in humans. To determine how P. aeruginosa affects the physiology of C. albicans and vice versa, we compared the proteomes of each species in mixed biofilms versus single-species biofilms. In addition, extracellular proteins were analyzed. We observed that, in mixed biofilms, both species showed differential expression of virulence proteins, multidrug resistance-associated proteins, proteases and cell defense, stress and iron-regulated proteins. Furthermore, in mixed biofilms, both species displayed an increase in mutability compared with monospecific biofilms. This characteristic was correlated with the downregulation of enzymes conferring protection against DNA oxidation. In mixed biofilms, P. aeruginosa regulates its production of various molecules involved in quorum sensing and induces the production of virulence factors (pyoverdine, rhamnolipids and pyocyanin), which are major contributors to the ability of this bacterium to cause disease. Overall, our results indicate that interspecies competition between these opportunistic pathogens enhances the production of virulence factors and increases mutability and thus can alter the course of host-pathogen interactions in polymicrobial infections. PMID:24739628

  20. Interspecies competition triggers virulence and mutability in Candida albicans-Pseudomonas aeruginosa mixed biofilms.

    PubMed

    Trejo-Hernández, Abigail; Andrade-Domínguez, Andrés; Hernández, Magdalena; Encarnación, Sergio

    2014-10-01

    Inter-kingdom and interspecies interactions are ubiquitous in nature and are important for the survival of species and ecological balance. The investigation of microbe-microbe interactions is essential for understanding the in vivo activities of commensal and pathogenic microorganisms. Candida albicans, a polymorphic fungus, and Pseudomonas aeruginosa, a Gram-negative bacterium, are two opportunistic pathogens that interact in various polymicrobial infections in humans. To determine how P. aeruginosa affects the physiology of C. albicans and vice versa, we compared the proteomes of each species in mixed biofilms versus single-species biofilms. In addition, extracellular proteins were analyzed. We observed that, in mixed biofilms, both species showed differential expression of virulence proteins, multidrug resistance-associated proteins, proteases and cell defense, stress and iron-regulated proteins. Furthermore, in mixed biofilms, both species displayed an increase in mutability compared with monospecific biofilms. This characteristic was correlated with the downregulation of enzymes conferring protection against DNA oxidation. In mixed biofilms, P. aeruginosa regulates its production of various molecules involved in quorum sensing and induces the production of virulence factors (pyoverdine, rhamnolipids and pyocyanin), which are major contributors to the ability of this bacterium to cause disease. Overall, our results indicate that interspecies competition between these opportunistic pathogens enhances the production of virulence factors and increases mutability and thus can alter the course of host-pathogen interactions in polymicrobial infections. PMID:24739628

  1. Biofilm Formation and β-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa

    PubMed Central

    Heydari, Samira; Eftekhar, Fereshteh

    2015-01-01

    Background: Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum β-lactamases (ESBL), metallo β-lactamases (MBL), and AmpC β-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster. Objectives: The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC β-lactamase production with biofilm formation among the isolates. Materials and Methods: Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR. Results: Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm. Conclusions: Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC

  2. The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance of Pseudomonas aeruginosa

    PubMed Central

    Oglesby-Sherrouse, Amanda G.; Djapgne, Louise; Nguyen, Angela T.; Vasil, Adriana I.; Vasil, Michael L.

    2014-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment of such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, non-siderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by sub-inhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development. PMID:24436170

  3. Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin

    PubMed Central

    Das, Manash C.; Sandhu, Padmani; Gupta, Priya; Rudrapaul, Prasenjit; De, Utpal C.; Tribedi, Prosun; Akhter, Yusuf; Bhattacharjee, Surajit

    2016-01-01

    Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It’s antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis. PMID:27000525

  4. Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin

    NASA Astrophysics Data System (ADS)

    Das, Manash C.; Sandhu, Padmani; Gupta, Priya; Rudrapaul, Prasenjit; de, Utpal C.; Tribedi, Prosun; Akhter, Yusuf; Bhattacharjee, Surajit

    2016-03-01

    Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It’s antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis.

  5. Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin.

    PubMed

    Das, Manash C; Sandhu, Padmani; Gupta, Priya; Rudrapaul, Prasenjit; De, Utpal C; Tribedi, Prosun; Akhter, Yusuf; Bhattacharjee, Surajit

    2016-01-01

    Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It's antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis. PMID:27000525

  6. Impairment of Pseudomonas aeruginosa Biofilm Resistance to Antibiotics by Combining the Drugs with a New Quorum-Sensing Inhibitor

    PubMed Central

    Lajoie, Barbora; El Hage, Salome; Baziard, Genevieve; Roques, Christine

    2015-01-01

    Pseudomonas aeruginosa plays an important role in chronic lung infections among patients with cystic fibrosis (CF) through its ability to form antibiotic-resistant biofilms. In P. aeruginosa, biofilm development and the production of several virulence factors are mainly regulated by the rhl and las quorum-sensing (QS) systems, which are controlled by two N-acyl-homoserine lactone signal molecules. In a previous study, we discovered an original QS inhibitor, N-(2-pyrimidyl)butanamide, called C11, based on the structure of C4-homoserine lactone, and found that it is able to significantly inhibit P. aeruginosa biofilm formation. However, recent data indicate that P. aeruginosa grows under anaerobic conditions and forms biofilms in the lungs of CF patients that are denser and more robust than those formed under aerobic conditions. Our confocal microscopy observations of P. aeruginosa biofilms developed under aerobic and anaerobic conditions confirmed that the biofilms formed under these two conditions have radically different architectures. C11 showed significant dose-dependent antibiofilm activity on biofilms grown under both aerobic and anaerobic conditions, with a greater inhibitory effect being seen under conditions of anaerobiosis. Gene expression analyses performed by quantitative reverse transcriptase PCR showed that C11 led to the significant downregulation of rhl QS regulatory genes but also to the downregulation of both las QS regulatory genes and QS system-regulated virulence genes, rhlA and lasB. Furthermore, the activity of C11 in combination with antibiotics against P. aeruginosa biofilms was tested, and synergistic antibiofilm activity between C11 and ciprofloxacin, tobramycin, and colistin was obtained under both aerobic and anaerobic conditions. This study demonstrates that C11 may increase the efficacy of treatments for P. aeruginosa infections by increasing the susceptibility of biofilms to antibiotics and by attenuating the pathogenicity of the

  7. Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms

    PubMed Central

    Chua, Song Lin; Yam, Joey Kuok Hoong; Hao, Piliang; Adav, Sunil S.; Salido, May Margarette; Liu, Yang; Givskov, Michael; Sze, Siu Kwan; Tolker-Nielsen, Tim; Yang, Liang

    2016-01-01

    Drug resistance and tolerance greatly diminish the therapeutic potential of antibiotics against pathogens. Antibiotic tolerance by bacterial biofilms often leads to persistent infections, but its mechanisms are unclear. Here we use a proteomics approach, pulsed stable isotope labelling with amino acids (pulsed-SILAC), to quantify newly expressed proteins in colistin-tolerant subpopulations of Pseudomonas aeruginosa biofilms (colistin is a ‘last-resort' antibiotic against multidrug-resistant Gram-negative pathogens). Migration is essential for the formation of colistin-tolerant biofilm subpopulations, with colistin-tolerant cells using type IV pili to migrate onto the top of the colistin-killed biofilm. The colistin-tolerant cells employ quorum sensing (QS) to initiate the formation of new colistin-tolerant subpopulations, highlighting multicellular behaviour in antibiotic tolerance development. The macrolide erythromycin, which has been previously shown to inhibit the motility and QS of P. aeruginosa, boosts biofilm eradication by colistin. Our work provides insights on the mechanisms underlying the formation of antibiotic-tolerant populations in bacterial biofilms and indicates research avenues for designing more efficient treatments against biofilm-associated infections. PMID:26892159

  8. Insulin Treatment Modulates the Host Immune System To Enhance Pseudomonas aeruginosa Wound Biofilms

    PubMed Central

    Watters, Chase; Everett, Jake A.; Haley, Cecily; Clinton, Allie

    2014-01-01

    Diabetes affects 25.8 million people in the United States, or 8.3% of the population, and these numbers are even higher in developing countries. Diabetic patients are more susceptible to the development of chronic wounds with debilitating bacterial infections than nondiabetics. Previously, we compared the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause biofilm-associated infections in chronic wounds of diabetic and nondiabetic mice (C. Watters, K. DeLeon, U. Trivedi, J. A. Griswold, M. Lyte, K. J. Hampel, M. J. Wargo, and K. P. Rumbaugh, Med. Microbiol. Immunol. 202:131–141, 2013). Unexpectedly, we observed that insulin-treated diabetic mice had significantly more biofilm in their wounds, which correlated with higher antibiotic tolerance. Here, we investigated whether insulin treatment modulates the diabetic immune system to favor P. aeruginosa biofilm formation. Utilizing a murine chronic wound model, we found that DNA protected P. aeruginosa in the wounds of insulin-treated diabetic mice from antibiotic treatment. We also observed increased numbers of neutrophils, reduced numbers of macrophages, and increased cell death in the wounds of diabetic mice on insulin therapy. Taken together, these data suggest that high levels of lysed neutrophils in the wounds of diabetic mice on insulin, combined with fewer macrophages to remove the cellular debris, contribute to increased DNA levels, which enhance P. aeruginosa biofilms. PMID:24126517

  9. Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination

    PubMed Central

    Anderson, Gregory G.; Kenney, Thomas F.; MacLeod, David L.; Henig, Noreen R.; O’Toole, George A.

    2016-01-01

    Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4 : 1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L−1, while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L−1 FTI and 16 mg L−1 tobramycin. Fosfomycin (1024 mg L−1) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L−1) and tobramycin (4 mg L−1) and high concentrations of fosfomycin (1024 mg L−1) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides. PMID:23620118

  10. Efficacy of ciprofloxacin-clarithromycin combination against drug-resistant Pseudomonas aeruginosa mature biofilm using in vitro experimental model.

    PubMed

    Elkhatib, Walid; Noreddin, Ayman

    2014-12-01

    Pseudomonas aeruginosa is the main cause of mortality in cystic fibrosis patients and eradication of its biofilm represents a substantial problem clinically. In this study, biofilm of a cystic fibrosis strain P. aeruginosa PACI22 was established and confocal laser scanning microscopy was utilized for biofilm visualization. A quantitative time-kill biofilm model was implemented in vitro to assess the biocidal effect of ciprofloxacin, clarithromycin, and their combination at concentration levels ranged from 0.5× to 64× minimum biofilm inhibitory concentrations (MBIC) against the biofilm and the mean log bacterial densities (Log CFU/ml) retrieved from the biofilm were monitored by frequent sampling at 0, 3, 6, 9, 12, and 24 hr throughout the experiment. The results revealed that none of the tested antibiotics alone could completely eradicate the biofilm-ensconced bacteria at 0.5-64× MBIC values after 24 hr of treatment. Conversely, ciprofloxacin-clarithromycin combination at 32-64× MBIC entirely exterminated the biofilm. Furthermore, a substantial in vitro synergism between ciprofloxacin and clarithromycin against the biofilm was experimentally verified. This promising synergism affords scientific rationale for further in vivo investigations to evaluate the therapeutic potential of this combination for treatment of chronic pulmonary infections caused by P. aeruginosa biofilms. PMID:25050970

  11. Pseudomonas aeruginosa Promotes Escherichia coli Biofilm Formation in Nutrient-Limited Medium

    PubMed Central

    Culotti, Alessandro; Packman, Aaron I.

    2014-01-01

    Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions. PMID:25198725

  12. Pseudomonas aeruginosa promotes Escherichia coli biofilm formation in nutrient-limited medium.

    PubMed

    Culotti, Alessandro; Packman, Aaron I

    2014-01-01

    Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions. PMID:25198725

  13. Subinhibitory concentration of ciprofloxacin targets quorum sensing system of Pseudomonas aeruginosa causing inhibition of biofilm formation & reduction of virulence

    PubMed Central

    Gupta, Parul; Chhibber, Sanjay; Harjai, Kusum

    2016-01-01

    Background & objectives: Biofilms formed by Pseudomonas aeruginosa lead to persistent infections. Use of antibiotics for the treatment of biofilm induced infection poses a threat towards development of resistance. Therefore, the research is directed towards exploring the property of antibiotics which may alter the virulence of an organism besides altering its growth. The aim of this study was to evaluate the role of subinhibitory concentration of ciprofloxacin (CIP) in inhibiting biofilm formation and virulence of P. aeruginosa. Methods: Antibiofilm potential of subinhibitory concentration of CIP was evaluated in terms of log reduction, biofilm forming capacity and coverslip assay. P. aeruginosa isolates (grown in the presence and absence of sub-MIC of CIP) were also evaluated for inhibition in motility, virulence factor production and quorum sensing (QS) signal production. Results: Sub-minimum inhibitory concentration (sub-MIC) of CIP significantly reduced the motility of P. aeruginosa stand and strain and clinical isolates and affected biofilm forming capacity. Production of protease, elastase, siderophore, alginate, and rhamnolipid was also significantly reduced by CIP. Interpretation & conclusions: Reduction in virulence factors and biofilm formation was due to inhibition of QS mechanism which was indicated by reduced production of QS signal molecules by P. aeruginosa in presence of subinhibitory concentration of CIP. PMID:27488009

  14. A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses

    PubMed Central

    2010-01-01

    Background Pseudomonas aeruginosa is commonly associated with contact lens (CL) -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented. Results In the current investigation, a novel in-vitro biofilm model for studying the adherence of P. aeruginosa to hydrogel CLs was established. Nutritional and interfacial conditions similar to those in the eye of a CL wearer were created through the involvement of a solid:liquid and a solid:air interface, shear forces and a complex artificial tear fluid. Bioburdens varied depending on the CL material and biofilm maturation occurred after 72 h incubation. Whilst a range of biofilm morphologies were visualised including dispersed and adherent bacterial cells, aggregates and colonies embedded in extracellular polymer substances (EPS), EPS fibres, mushroom-like formations, and crystalline structures, a compact and heterogeneous biofilm morphology predominated on all CL materials. Conclusions In order to better understand the process of biofilm formation on CLs and to test the efficacy of CL care solutions, representative in-vitro biofilm models are required. Here, we present a three-phase biofilm model that simulates the environment in the eye of a CL wearer and thus generates biofilms which resemble those commonly observed in-situ. PMID:21062489

  15. Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines.

    PubMed

    Morales, Diana K; Grahl, Nora; Okegbe, Chinweike; Dietrich, Lars E P; Jacobs, Nicholas J; Hogan, Deborah A

    2013-01-01

    Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease. IMPORTANCE Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the

  16. Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.

    PubMed

    Herbst, Florian-Alexander; Søndergaard, Mads T; Kjeldal, Henrik; Stensballe, Allan; Nielsen, Per H; Dueholm, Morten S

    2015-01-01

    The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid-mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, which turned P. aeruginosa PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids on the physiology of P. aeruginosa with subsequent implications for biofilm formation and chronic infections. PMID:25317949

  17. The Pseudomonas aeruginosa Type III Translocon Is Required for Biofilm Formation at the Epithelial Barrier

    PubMed Central

    Tran, Cindy S.; Rangel, Stephanie M.; Almblad, Henrik; Kierbel, Arlinet; Givskov, Michael; Tolker-Nielsen, Tim; Hauser, Alan R.; Engel, Joanne N.

    2014-01-01

    Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection. PMID:25375398

  18. The Pseudomonas aeruginosa type III translocon is required for biofilm formation at the epithelial barrier.

    PubMed

    Tran, Cindy S; Rangel, Stephanie M; Almblad, Henrik; Kierbel, Arlinet; Givskov, Michael; Tolker-Nielsen, Tim; Hauser, Alan R; Engel, Joanne N

    2014-11-01

    Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection. PMID:25375398

  19. Decrease of Pseudomonas aeruginosa biofilm formation by food waste materials.

    PubMed

    Maderova, Zdenka; Horska, Katerina; Kim, Sang-Ryoung; Lee, Chung-Hak; Pospiskova, Kristyna; Safarikova, Mirka; Safarik, Ivo

    2016-01-01

    The formation of bacterial biofilm on various surfaces has significant negative economic effects. The aim of this study was to find a simple procedure to decrease the Pseudomonas aeruginosa biofilm formation in a water environment by using different food waste biological materials as signal molecule adsorbents. The selected biomaterials did not reduce the cell growth but affected biofilm formation. Promising biomaterials were magnetically modified in order to simplify manipulation and facilitate their magnetic separation. The best biocomposite, magnetically modified spent grain, exhibited substantial adsorption of signal molecules and decreased the biofilm formation. These results suggest that selected food waste materials and their magnetically responsive derivatives could be applied to solve biofilm problems in water environment. PMID:27148715

  20. Targeting iron uptake to control Pseudomonas aeruginosa infections in cystic fibrosis.

    PubMed

    Smith, Daniel J; Lamont, Iain L; Anderson, Greg J; Reid, David W

    2013-12-01

    The aerobic Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for life-threatening acute and chronic infections in humans. As part of chronic infection P. aeruginosa forms biofilms, which shield the encased bacteria from host immune clearance and provide an impermeable and protective barrier against currently available antimicrobial agents. P. aeruginosa has an absolute requirement for iron for infection success. By influencing cell-cell communication (quorum sensing) and virulence factor expression, iron is a powerful regulator of P. aeruginosa behaviour. Consequently, the imposed perturbation of iron acquisition systems has been proposed as a novel therapeutic approach to the treatment of P. aeruginosa biofilm infection. In this review, we explore the influence of iron availability on P. aeruginosa infection in the lungs of the people with the autosomal recessive condition cystic fibrosis as an archetypal model of chronic P. aeruginosa biofilm infection. Novel therapeutics aimed at disrupting P. aeruginosa are discussed, with an emphasis placed on identifying the barriers that need to be overcome in order to translate these promising in vitro agents into effective therapies in human pulmonary infections. PMID:23143541

  1. Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma

    PubMed Central

    Alkawareek, Mahmoud Y.; Algwari, Qais Th.; Laverty, Garry; Gorman, Sean P.; Graham, William G.; O'Connell, Deborah; Gilmore, Brendan F.

    2012-01-01

    Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (∼10′s s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity. PMID

  2. Biofilms in periprosthetic orthopedic infections

    PubMed Central

    McConoughey, Stephen J; Howlin, Rob; Granger, Jeff F; Manring, Maurice M; Calhoun, Jason H; Shirtlif, Mark; Kathju, Sandeep; Stoodley, Paul

    2015-01-01

    As the number of total joint arthroplasty and internal fixation procedures continues to rise, the threat of infection following surgery has significant clinical implications. These infections may have highly morbid consequences to patients, who often endure additional surgeries and lengthy exposures to systemic antibiotics, neither of which are guaranteed to resolve the infection. Of particular concern is the threat of bacterial biofilm development, since biofilm-mediated infections are difficult to diagnose and effective treatments are lacking. Developing therapeutic strategies have targeted mechanisms of biofilm formation and the means by which these bacteria communicate with each other to take on specialized roles such as persister cells within the biofilm. In addition, prevention of infection through novel coatings for prostheses and the local delivery of high concentrations of antibiotics by absorbable carriers has shown promise in laboratory and animal studies. Biofilm development, especially in an arthoplasty environment, and future diagnostic and treatment options are discussed. PMID:25302955

  3. Distribution and Inhibition of Liposomes on Staphylococcus aureus and Pseudomonas aeruginosa Biofilm

    PubMed Central

    Dong, Dong; Thomas, Nicky; Thierry, Benjamin; Vreugde, Sarah; Prestidge, Clive A.; Wormald, Peter-John

    2015-01-01

    Background Staphylococcus aureus and Pseudomonas aeruginosa are major pathogens in chronic rhinosinusitis (CRS) and their biofilms have been associated with poorer postsurgical outcomes. This study investigated the distribution and anti-biofilm effect of cationic (+) and anionic (-) phospholipid liposomes with different sizes (unilamellar and multilamellar vesicle, ULV and MLV respectively) on S. aureus and P. aeruginosa biofilms. Method Specific biofilm models for S. aureus ATCC 25923 and P. aeruginosa ATCC 15692 were established. Liposomal distribution was determined by observing SYTO9 stained biofilm exposed to DiI labeled liposomes using confocal scanning laser microscopy, followed by quantitative image analysis. The anti-biofilm efficacy study was carried out by using the alamarBlue assay to test the relative viability of biofilm treated with various liposomes for 24 hours and five minutes. Results The smaller ULVs penetrated better than larger MLVs in both S. aureus and P. aeruginosa biofilm. Except that +ULV and –ULV displayed similar distribution in S. aureus biofilm, the cationic liposomes adhered better than their anionic counterparts. Biofilm growth was inhibited at 24-hour and five-minute exposure time, although the decrease of viability for P. aeruginosa biofilm after liposomal treatment did not reach statistical significance. Conclusion The distribution and anti-biofilm effects of cationic and anionic liposomes of different sizes differed in S. aureus and P. aeruginosa biofilms. Reducing the liposome size and formulating liposomes as positively charged enhanced the penetration and inhibition of S. aureus and P. aeruginosa biofilms. PMID:26125555

  4. Pseudomonas aeruginosa facilitates Campylobacter jejuni growth in biofilms under oxic flow conditions.

    PubMed

    Culotti, Alessandro; Packman, Aaron I

    2015-12-01

    We investigated the growth of Campylobacter jejuni in biofilms with Pseudomonas aeruginosa under oxic flow conditions. We observed the growth of C. jejuni in mono-culture, deposited on pre-established P. aeruginosa biofilms, and co-inoculated with P. aeruginosa. In mono-culture, C. jejuni was unable to form biofilms. However, deposited C. jejuni continuously grew on pre-established P. aeruginosa biofilms for a period of 3 days. The growth of scattered C. jejuni clusters was strictly limited to the P. aeruginosa biofilm surface, and no intergrowth was observed. Co-culturing of C. jejuni and P. aeruginosa also enabled the growth of both organisms in biofilms, with C. jejuni clusters developing on the surface of the P. aeruginosa biofilm. Dissolved oxygen (DO) measurements in the medium showed that P. aeruginosa biofilms depleted the effluent DO from 9.0 to 0.5 mg L(-1) 24 hours after inoculation. The localized microaerophilic environment generated by P. aeruginosa promoted the persistence and growth of C. jejuni. Our findings show that P. aeruginosa not only prolongs the survival of C. jejuni under oxic conditions, but also enables the growth of C. jejuni on the surface of P. aeruginosa biofilms. PMID:26610432

  5. Mannitol Enhances Antibiotic Sensitivity of Persister Bacteria in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Barraud, Nicolas; Buson, Alberto; Jarolimek, Wolfgang; Rice, Scott A.

    2013-01-01

    The failure of antibiotic therapies to clear Pseudomonas aeruginosa lung infection, the key mortality factor for cystic fibrosis (CF) patients, is partly attributed to the high tolerance of P. aeruginosa biofilms. Mannitol has previously been found to restore aminoglycoside sensitivity in Escherichia coli by generating a proton-motive force (PMF), suggesting a potential new strategy to improve antibiotic therapy and reduce disease progression in CF. Here, we used the commonly prescribed aminoglycoside tobramycin to select for P. aeruginosa persister cells during biofilm growth. Incubation with mannitol (10–40 mM) increased tobramycin sensitivity of persister cells up to 1,000-fold. Addition of mannitol to pre-grown biofilms was able to revert the persister phenotype and improve the efficacy of tobramycin. This effect was blocked by the addition of a PMF inhibitor or in a P. aeruginosa mutant strain unable to metabolise mannitol. Addition of glucose and NaCl at high osmolarity also improved the efficacy of tobramycin although to a lesser extent compared to mannitol. Therefore, the primary effect of mannitol in reverting biofilm associated persister cells appears to be an active, physiological response, associated with a minor contribution of osmotic stress. Mannitol was tested against clinically relevant strains, showing that biofilms containing a subpopulation of persister cells are better killed in the presence of mannitol, but a clinical strain with a high resistance to tobramycin was not affected by mannitol. Overall, these results suggest that in addition to improvements in lung function by facilitating mucus clearance in CF, mannitol also affects antibiotic sensitivity in biofilms and does so through an active, physiological response. PMID:24349568

  6. Biofilm formation by Staphylococcus epidermidis on peritoneal dialysis catheters and the effects of extracellular products from Pseudomonas aeruginosa.

    PubMed

    Pihl, Maria; Arvidsson, Anna; Skepö, Marie; Nilsson, Martin; Givskov, Michael; Tolker-Nielsen, Tim; Svensäter, Gunnel; Davies, Julia R

    2013-04-01

    Biofilm formation by Staphylococcus epidermidis is a cause of infections related to peritoneal dialysis (PD). We have used a PD catheter flow-cell model in combination with confocal scanning laser microscopy and atomic force microscopy to study biofilm formation by S. epidermidis. Adherence to serum-coated catheters was four times greater than to uncoated ones, suggesting that S. epidermidis binds to serum proteins on the catheter surface. Pseudomonas aeruginosa biofilm supernatant interfered with the formation of a serum protein coat thereby reducing the capacity for biofilm formation in S. epidermidis. Supernatants from ΔpelA, ΔpslBCD and ΔrhlAB strains of P. aeruginosa showed no differences from the wild-type supernatant indicating that the effect on serum coat formation was not due to rhamnolipids or the PelA and PslBCD polysaccharides. Supernatant from P. aeruginosa also dispersed established S. epidermidis biofilms. Supernatants lacking PelA or PslBCD showed no differences from the wild type but that from a ΔrhlAB strain, showed reduced, but not abolished, capacity for dispersal. This suggests that rhamnolipids are involved but not wholly responsible for the effect. Thus, supernatants from P. aeruginosa contain promising substances for the prevention and treatment of biofilm infections, although further work is required to identity more active components. PMID:23620182

  7. Anti-Biofilm Activities from Marine Cold Adapted Bacteria Against Staphylococci and Pseudomonas aeruginosa

    PubMed Central

    Papa, Rosanna; Selan, Laura; Parrilli, Ermenegilda; Tilotta, Marco; Sannino, Filomena; Feller, Georges; Tutino, Maria L.; Artini, Marco

    2015-01-01

    Microbial biofilms have great negative impacts on the world’s economy and pose serious problems to industry, public health and medicine. The interest in the development of new approaches for the prevention and treatment of bacterial adhesion and biofilm formation has increased. Since, bacterial pathogens living in biofilm induce persistent chronic infections due to the resistance to antibiotics and host immune system. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the appearance of resistant mutants. Many bacteria secrete anti-biofilm molecules that function in regulating biofilm architecture or mediating the release of cells from it during the dispersal stage of biofilm life cycle. Cold-adapted marine bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. The anti-biofilm activity of cell-free supernatants derived from sessile and planktonic cultures of cold-adapted bacteria belonging to Pseudoalteromonas, Psychrobacter, and Psychromonas species were tested against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa strains. Reported results demonstrate that we have selected supernatants, from cold-adapted marine bacteria, containing non-biocidal agents able to destabilize biofilm matrix of all tested pathogens without killing cells. A preliminary physico-chemical characterization of supernatants was also performed, and these analyses highlighted the presence of molecules of different nature that act by inhibiting biofilm formation. Some of them are also able to impair the initial attachment of the bacterial cells to the surface, thus likely containing molecules acting as anti-biofilm surfactant molecules. The described ability of cold-adapted bacteria to produce effective anti-biofilm molecules paves the way to further characterization of the most promising molecules and to test their

  8. The MerR-Like Transcriptional Regulator BrlR Contributes to Pseudomonas aeruginosa Biofilm Tolerance

    PubMed Central

    Liao, Julie

    2012-01-01

    Biofilms are composed of surface-attached microbial communities. A hallmark of biofilms is their profound tolerance of antimicrobial agents. While biofilm drug tolerance has been considered to be multifactorial, our findings indicate, instead, that bacteria within biofilms employ a classical regulatory mechanism to resist the action of antimicrobial agents. Here we report that the transcriptional regulator BrlR, a member of the MerR family of multidrug transport activators, plays a role in the high-level drug tolerance of biofilms formed by Pseudomonas aeruginosa. Expression of brlR was found to be biofilm specific, with brlR inactivation not affecting biofilm formation, motility, or pslA expression but increasing ndvB expression. Inactivation of brlR rendered biofilms but not planktonic cells grown to exponential or stationary phase significantly more susceptible to hydrogen peroxide and five different classes of antibiotics by affecting the MICs and the recalcitrance of biofilms to killing by microbicidal antimicrobial agents. In contrast, overexpression of brlR rendered both biofilms and planktonic cells more tolerant to the same compounds. brlR expression in three cystic fibrosis (CF) isolates was elevated regardless of the mode of growth, suggesting a selection for constitutive brlR expression upon in vivo biofilm formation associated with chronic infections. Despite increased brlR expression, however, isolate CF1-8 was as susceptible to tobramycin as was a ΔbrlR mutant because of a nonsense mutation in brlR. Our results indicate for the first time that biofilms employ a specific regulatory mechanism to resist the action of antimicrobial agents in a BrlR-dependent manner which affects MIC and recalcitrance to killing by microbicidal antimicrobial agents. PMID:22730129

  9. ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Cho, Moo Hwan; Lee, Jintae

    2014-12-01

    The opportunistic pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilms of this bacterium are much more resistant to antibiotics than planktonic cells. Thirty-six metal ions have been investigated to identify antivirulence and antibiofilm metal ions. Zinc ions and ZnO nanoparticles were found to markedly inhibit biofilm formation and the production of pyocyanin, Pseudomonas quinolone signal (PQS), pyochelin, and hemolytic activity of P. aeruginosa without affecting the growth of planktonic cells. Transcriptome analyses showed that ZnO nanoparticles induce the zinc cation efflux pump czc operon and several important transcriptional regulators (porin gene opdT and type III repressor ptrA), but repress the pyocyanin-related phz operon, which explains observed phenotypic changes. A mutant study showed that the effects of ZnO nanoparticles on the control of pyocyanin production and biofilm formation require the czc regulator CzcR. In addition, ZnO nanoparticles markedly increased the cellular hydrophilicity of P. aeruginosa cells. Our results support that ZnO nanoparticles are potential antivirulence materials against recalcitrant P. aeruginosa infections and possibly other important pathogens. PMID:24958247

  10. Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

    PubMed

    Bandara, H M H N; K Cheung, B P; Watt, R M; Jin, L J; Samaranayake, L P

    2013-02-01

    Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation. PMID:23194472

  11. Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance

    PubMed Central

    Periasamy, Saravanan; Nair, Harikrishnan A. S.; Lee, Kai W. K.; Ong, Jolene; Goh, Jie Q. J.; Kjelleberg, Staffan; Rice, Scott A.

    2015-01-01

    Pseudomonas aeruginosa PAO1 produces three polysaccharides, alginate, Psl, and Pel that play distinct roles in attachment and biofilm formation for monospecies biofilms. Considerably less is known about their role in the development of mixed species biofilm communities. This study has investigated the roles of alginate, Psl, and Pel during biofilm formation of P. aeruginosa in a defined and experimentally informative mixed species biofilm community, consisting of P. aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Loss of the Psl polysaccharide had the biggest impact on the integration of P. aeruginosa in the mixed species biofilms, where the percent composition of the psl mutant was significantly lower (0.06%) than its wild-type (WT) parent (2.44%). In contrast, loss of the Pel polysaccharide had no impact on mixed species biofilm development. Loss of alginate or its overproduction resulted in P. aeruginosa representing 8.4 and 18.11%, respectively, of the mixed species biofilm. Dual species biofilms of P. aeruginosa and K. pneumoniae were not affected by loss of alginate, Pel, or Psl, while the mucoid P. aeruginosa strain achieved a greater biomass than its parent strain. When P. aeruginosa was grown with P. protegens, loss of the Pel or alginate polysaccharides resulted in biofilms that were not significantly different from biofilms formed by the WT PAO1. In contrast, overproduction of alginate resulted in biofilms that were comprised of 35–40% of P. aeruginosa, which was significantly higher than the WT (5–20%). Loss of the Psl polysaccharide significantly reduced the percentage composition of P. aeruginosa in dual species biofilms with P. protegens (<1%). Loss of the Psl polysaccharide significantly disrupted the communal stress resistance of the three species biofilms. Thus, the polysaccharide composition of an individual species significantly impacts mixed species biofilm development and the emergent properties of such communities. PMID

  12. Disruption of Contact Lens–Associated Pseudomonas aeruginosa Biofilms Formed in the Presence of Neutrophils

    PubMed Central

    Parks, Quinn M.; Young, Robert L.; Kret, Jennifer; Poch, Katie R.; Malcolm, Kenneth C.; Nichols, David P.; Nichols, Michelle; Zhu, Meifang; Cavanagh, H. Dwight; Nick, Jerry A.

    2011-01-01

    Purpose. To evaluate the capacity of neutrophils to enhance biofilm formation on contact lenses by an infectious Pseudomonas aeruginosa (PA) corneal isolate. Agents that target F-actin and DNA were tested as a therapeutic strategy for disrupting biofilms formed in the setting of neutrophils in vitro and for limiting the infectious bioburden in vivo. Methods. Biofilm formation by infectious PA strain 6294 was assessed in the presence of neutrophils on a static biofilm plate and on unworn etafilcon A soft contact lenses. A d-isomer of poly(aspartic acid) was used alone and with DNase to reduce biofilm formation on test contact lenses. The gentamicin survival assay was used to determine the effectiveness of the test compound in reducing subsequent intracellular bacterial load in the corneal epithelium in a contact lens infection model in the rabbit. Results. In a static reactor and on hydrogel lenses, PA biofilm density was enhanced 30-fold at 24 hours in the presence of neutrophils (P < 0.0001). The combination of DNase and anionic poly(aspartic acid) reduced the PA biofilms formed in the presence of activated neutrophils by 79.2% on hydrogel contact lenses (P < 0.001). An identical treatment resulted in a 41% reduction in internalized PA in the rabbit corneal epithelium after 24 hours (P = 0.03). Conclusions. These results demonstrate that PA can exploit the presence of neutrophils to form biofilm on contact lenses within a short time. Incorporation of F-actin and DNA represent a mechanism for neutrophil-induced biofilm enhancement and are targets for available agents to disrupt pathogenic biofilms formed on contact lenses and as a treatment for established corneal infections. PMID:21245396

  13. Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation

    PubMed Central

    Rosay, Thibaut; Bazire, Alexis; Diaz, Suraya; Clamens, Thomas; Blier, Anne-Sophie; Mijouin, Lily; Hoffmann, Brice; Sergent, Jacques-Aurélien; Bouffartigues, Emeline; Boireau, Wilfrid; Vieillard, Julien; Hulen, Christian; Dufour, Alain; Harmer, Nicholas J.; Feuilloley, Marc G. J.

    2015-01-01

    ABSTRACT Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression. PMID:26307165

  14. Novel Strategies for the Treatment of Pseudomonas aeruginosa Infections.

    PubMed

    Wagner, Stefanie; Sommer, Roman; Hinsberger, Stefan; Lu, Cenbin; Hartmann, Rolf W; Empting, Martin; Titz, Alexander

    2016-07-14

    Infections with Pseudomonas aeruginosa have become a concerning threat in hospital-acquired infections and for cystic fibrosis patients. The major problem leading to high mortality lies in the appearance of drug-resistant strains. Therefore, a vast number of approaches to develop novel anti-infectives is currently pursued. These diverse strategies span from killing (new antibiotics) to disarming (antivirulence) the pathogen. Particular emphasis lies on the development of compounds that inhibit biofilms formed in chronic infections to restore susceptibility toward antibiotics. Numerous promising results are summarized in this perspective. Antibiotics with a novel mode of action will be needed to avoid cross resistance against currently used therapeutic agents. Importantly, antivirulence drugs are expected to yield a significantly reduced rate of resistance development. Most developments are still far from the application. It can however be expected that combination therapies, also containing antivirulence agents, will pave the way toward novel treatment options against P. aeruginosa. PMID:26804741

  15. TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    Background Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult to treat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections and broad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified to modulate virulence in enteric Gram-negative pathogens. Results Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocytic amoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cell attachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, of important virulence-related genes such as those involved in regulation and assembly of the Type III secretion system, consistent with the observed phenotypes and role in virulence of P. aeruginosa. Conclusions These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and is involved in multiple virulence-related characteristics. PMID:23570569

  16. Evaluation of the ability of C. albicans to form biofilm in the presence of phage-resistant phenotypes of P. aeruginosa.

    PubMed

    Pires, Diana P; Silva, Sónia; Almeida, Carina; Henriques, Mariana; Anderson, Erin M; Lam, Joseph S; Sillankorva, Sanna; Azeredo, Joana

    2013-01-01

    Pseudomonas aeruginosa and Candida albicans are disparate microbial species, but both are known to be opportunistic pathogens frequently associated with nosocomial infections. The aim of this study was to provide a better understanding of the interactions between these microorganisms in dual-species biofilms. Several bacteriophage-resistant P. aeruginosa phenotypes have been isolated and were used in dual-species mixed-biofilm studies. Twenty-four and 48 h mixed-biofilms were formed using the isolated phenotypes of phage-resistant P. aeruginosa and these were compared with similar experiments using other P. aeruginosa strains with a defined lipopolysaccharide (LPS) deficiency based on chromosomal knockout of specific LPS biosynthetic genes. Overall, the results showed that the variants of phage-resistant P. aeruginosa and LPS mutants were both less effective in inhibiting the growth of C. albicans in mixed-biofilms compared to the wild-type strains of P. aeruginosa. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. In conclusion, the ability of strains of P. aeruginosa to inhibit the formation of a biofilm of C. albicans appears to be correlated with the LPS chain lengths of phenotypes of P. aeruginosa, suggesting that LPS has a suppressive effect on the growth of C. albicans. PMID:24063626

  17. Laser irradiation effect on Staphylococcus aureus and Pseudomonas aeruginosa biofilms isolated from venous leg ulcer.

    PubMed

    Baffoni, Marina; Bessa, Lucinda J; Grande, Rossella; Di Giulio, Mara; Mongelli, Matteo; Ciarelli, Antonio; Cellini, Luigina

    2012-10-01

    Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, represent a significant cause of morbidity in developed countries, predominantly in older patients. The aetiology of these wounds is probably multifactorial, but the role of bacteria in their pathogenesis is still unclear. Moreover, the presence of bacterial biofilms has been considered an important factor responsible for wounds chronicity. We aimed to investigate the laser action as a possible biofilm eradicating strategy, in order to attempt an additional treatment to antibiotic therapy to improve wound healing. In this work, the effect of near-infrared (NIR) laser was evaluated on mono and polymicrobial biofilms produced by two pathogenic bacterial strains, Staphylococcus aureus PECHA10 and Pseudomonas aeruginosa PECHA9, both isolated from a chronic venous leg ulcer. Laser effect was assessed by biomass measurement, colony forming unit count and cell viability assay. It was shown that the laser treatment has not affected the biofilms biomass neither the cell viability, although a small disruptive action was observed in the structure of all biofilms tested. A reduction on cell growth was observed in S. aureus and in polymicrobial biofilms. This work represents an initial in vitro approach to study the influence of NIR laser treatment on bacterial biofilms in order to explain its potentially advantageous effects in the healing process of chronic infected wounds. PMID:22182280

  18. Use of In-Biofilm Expression Technology To Identify Genes Involved in Pseudomonas aeruginosa Biofilm Development†

    PubMed Central

    Finelli, Antonio; Gallant, Claude V.; Jarvi, Keith; Burrows, Lori L.

    2003-01-01

    Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness. PMID:12700249

  19. Why Does the Healthy Cornea Resist Pseudomonas aeruginosa Infection?

    PubMed Central

    Evans, David J.; Fleiszig, Suzanne M. J.

    2013-01-01

    Purpose To provide our perspective on why the cornea is resistant to infection based on our research results with Pseudomonas aeruginosa. Perspective We focus on our current understanding of the interplay between bacteria, tear fluid and the corneal epithelium that determine health as the usual outcome, and propose a theoretical model for how contact lens wear might change those interactions to enable susceptibility to P. aeruginosa infection. Methods Use of “null-infection” in vivo models, cultured human corneal epithelial cells, contact lens-wearing animal models, and bacterial genetics help to elucidate mechanisms by which P. aeruginosa survive at the ocular surface, adheres, and traverses multilayered corneal epithelia. These models also help elucidate the molecular mechanisms of corneal epithelial innate defense. Results and Discussion Tear fluid and the corneal epithelium combine to make a formidable defense against P. aeruginosa infection of the cornea. Part of that defense involves the expression of antimicrobials such as β-defensins, the cathelicidin LL-37, cytokeratin-derived antimicrobial peptides, and RNase7. Immunomodulators such as SP-D and ST2 also contribute. Innate defenses of the cornea depend in part on MyD88, a key adaptor protein of TLR and IL-1R signaling, but the basal lamina represents the final barrier to bacterial penetration. Overcoming these defenses involves P. aeruginosa adaptation, expression of the type three secretion system, proteases, and P. aeruginosa biofilm formation on contact lenses. Conclusion After more than two decades of research focused on understanding how contact lens wear predisposes to P. aeruginosa infection, our working hypothesis places blame for microbial keratitis on bacterial adaptation to ocular surface defenses, combined with changes to the biochemistry of the corneal surface caused by trapping bacteria and tear fluid against the cornea under the lens. PMID:23601656

  20. Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation

    PubMed Central

    Biggs, Matthew B.; Papin, Jason A.

    2013-01-01

    Multiscale modeling is used to represent biological systems with increasing frequency and success. Multiscale models are often hybrids of different modeling frameworks and programming languages. We present the MATLAB-NetLogo extension (MatNet) as a novel tool for multiscale modeling. We demonstrate the utility of the tool with a multiscale model of Pseudomonas aeruginosa biofilm formation that incorporates both an agent-based model (ABM) and constraint-based metabolic modeling. The hybrid model correctly recapitulates oxygen-limited biofilm metabolic activity and predicts increased growth rate via anaerobic respiration with the addition of nitrate to the growth media. In addition, a genome-wide survey of metabolic mutants and biofilm formation exemplifies the powerful analyses that are enabled by this computational modeling tool. PMID:24147108

  1. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material.

    PubMed

    Vassena, Christian; Fenu, Simone; Giuliani, Francesco; Fantetti, Lia; Roncucci, Gabrio; Simonutti, Giulio; Romanò, Carlo Luca; De Francesco, Raffaele; Drago, Lorenzo

    2014-07-01

    Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms. PMID:24933446

  2. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

    PubMed

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants. PMID:25996839

  3. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

    PubMed Central

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants. PMID:25996839

  4. Dynamics of Mutator and Antibiotic-Resistant Populations in a Pharmacokinetic/Pharmacodynamic Model of Pseudomonas aeruginosa Biofilm Treatment ▿

    PubMed Central

    Macià, María D.; Pérez, José L.; Molin, Soeren; Oliver, Antonio

    2011-01-01

    Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 μg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstrated in situ for P. aeruginosa biofilms. PMID:21859941

  5. Vaccination against respiratory Pseudomonas aeruginosa infection

    PubMed Central

    Grimwood, Keith; Kyd, Jennelle M; Owen, Suzzanne J; Massa, Helen M; Cripps, Allan W

    2014-01-01

    Respiratory infections caused by Pseudomonas aeruginosa are a major clinical problem globally, particularly for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis (nCFB) and severe chronic obstructive pulmonary disease (COPD). In addition, critically ill and immunocompromised patients are also at significant risk of P. aeruginosa infection. For almost half a century, research efforts have focused toward development of a vaccine against infections caused by P. aeruginosa, but a licensed vaccine is not yet available. Significant advances in identifying potential vaccine antigens have been made. Immunisations via both the mucosal and systemic routes have been trialled in animal models and their effectiveness in clearing acute infections demonstrated. The challenge for translation of this research to human applications remains, since P. aeruginosa infections in the human respiratory tract can present both as an acute or chronic infection. In addition, immunisation prior to infection may not be possible for many patients with CF, nCFB or COPD. Therefore, development of a therapeutic vaccine provides an alternative approach for treatment of chronic infection. Preliminary animal and human studies suggest that mucosal immunisation may be effective as a therapeutic vaccine against P. aeruginosa respiratory infections. Nevertheless, more research is needed to improve our understanding of the basic biology of P. aeruginosa and the mechanisms needed to upregulate the induction of host immune pathways to prevent infection. Recognition of variability in the host immune responses for a range of patient health conditions at risk from P. aeruginosa infection is also required to support development of a successful vaccine delivery strategy and vaccine. Activation of mucosal immune responses may provide improved efficacy of vaccination for P. aeruginosa during both acute exacerbations and chronic infection. PMID:25483510

  6. Inhibition and dispersion of Pseudomonas aeruginosa biofilms by glycopeptide dendrimers targeting the fucose-specific lectin LecB.

    PubMed

    Johansson, Emma M V; Crusz, Shanika A; Kolomiets, Elena; Buts, Lieven; Kadam, Rameshwar U; Cacciarini, Martina; Bartels, Kai-Malte; Diggle, Stephen P; Cámara, Miguel; Williams, Paul; Loris, Remy; Nativi, Cristina; Rosenau, Frank; Jaeger, Karl-Erich; Darbre, Tamis; Reymond, Jean-Louis

    2008-12-22

    The human pathogenic bacterium Pseudomonas aeruginosa produces a fucose-specific lectin, LecB, implicated in tissue attachment and the formation of biofilms. To investigate if LecB inhibition disrupts these processes, high-affinity ligands were obtained by screening two 15,536-member combinatorial libraries of multivalent fucosyl-peptide dendrimers. The most potent LecB-ligands identified were dendrimers FD2 (C-Fuc-LysProLeu)(4)(LysPheLysIle)(2)LysHisIleNH(2) (IC(50) = 0.14 microM by ELLA) and PA8 (OFuc-LysAlaAsp)(4)(LysSerGlyAla)(2)LysHisIleNH(2) (IC(50) = 0.11 microM by ELLA). Dendrimer FD2 led to complete inhibition of P. aeruginosa biofilm formation (IC(50) approximately 10 microM) and induced complete dispersion of established biofilms in the wild-type strain and in several clinical P. aeruginosa isolates. These experiments suggest that LecB inhibition by high-affinity multivalent ligands could represent a therapeutic approach against P. aeruginosa infections by inhibition of biofilm formation and dispersion of established biofilms. PMID:19101469

  7. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics.

    PubMed

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the 'holy grail' in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  8. The Psl economy in early P. aeruginosa biofilm development

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Tseng, Boo Shan; Jin, Fan; Gibiansky, Max; Harrison, Joe; Parsek, Matthew; Wong, Gerard

    2012-02-01

    Psl from P. aeruginosa (PAO1) is a mannose- and galactose-rich exopolysaccharide (EPS). It has been shown that Psl plays an important role in bacterial surface adhesion. Here, we examine role of Psl in controlling motility and microcolony formation during early biofilm development, by translating video microscopy movies into searchable databases of bacterial trajectories. We use a massively-parallel cell tracking algorithm to extract the full motility history of every cell in a large community. We find that at early stages of growth, P. aeruginosa motility is guided by Psl and self-organize in a manner analogous to a capitalist economic system, resulting in a power law bacterial distribution where a small number of bacteria are extremely ``rich'' in communally produced Psl. By comparing overproducers and underproducers of Psl, we find that local Psl levels determine post-division cell fates: High local Psl levels drive the formation of sessile microcolonies that grow exponentially.

  9. d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

    PubMed Central

    Akers, Kevin S.; Romano, Desiree R.; Woodbury, Ronald L.; Hardy, Sharanda K.; Murray, Clinton K.; Wenke, Joseph C.

    2014-01-01

    Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of d-amino acids (d-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of d-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. d-Met, d-Phe, and d-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined with d-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of d-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of d-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity. PMID:24841260

  10. Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

    PubMed Central

    Alhede, Morten; Kragh, Kasper Nørskov; Qvortrup, Klaus; Allesen-Holm, Marie; van Gennip, Maria; Christensen, Louise D.; Jensen, Peter Østrup; Nielsen, Anne K.; Parsek, Matt; Wozniak, Dan; Molin, Søren; Tolker-Nielsen, Tim; Høiby, Niels; Givskov, Michael; Bjarnsholt, Thomas

    2011-01-01

    For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial

  11. Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

    PubMed Central

    Price, Katherine E.; Naimie, Amanda A.; Griffin, Edward F.; Bay, Charles

    2015-01-01

    ABSTRACT Cystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existing in vitro model of Pseudomonas aeruginosa biofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways. IMPORTANCE Deep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions

  12. Enzyme multilayer coatings inhibit Pseudomonas aeruginosa biofilm formation on urinary catheters.

    PubMed

    Ivanova, Kristina; Fernandes, Margarida M; Mendoza, Ernest; Tzanov, Tzanko

    2015-05-01

    Bacteria use a signaling mechanism called quorum sensing (QS) to form complex communities of surface-attached cells known as biofilms. This protective mode of growth allows them to resist antibiotic treatment and originates the majority of hospital-acquired infections. Emerging alternatives to control biofilm-associated infections and multidrug resistance development interfere with bacterial QS pathways, exerting less selective pressure on bacterial population. In this study, biologically stable coatings comprising the QS disrupting enzyme acylase were built on silicone urinary catheters using a layer-by-layer technique. This was achieved by the alternate deposition of negatively charged enzyme and positively charged polyethylenimine. The acylase-coated catheters efficiently quenched the QS in the biosensor strain Chromobacterium violaceum CECT 5999, demonstrated by approximately 50% inhibition of violacein production. These enzyme multilayer coatings significantly reduced the Pseudomonas aeruginosa ATCC 10145 biofilm formation under static and dynamic conditions in an in vitro catheterized bladder model. The quorum quenching enzyme coatings did not affect the viability of the human fibroblasts (BJ-5ta) over 7 days, corresponding to the extended useful life of urinary catheters. Such enzyme-based approach could be an alternative to the conventional antibiotic treatment for prevention of biofilm-associated urinary tract infections. PMID:25582561

  13. Strategies for combating bacterial biofilm infections

    PubMed Central

    Wu, Hong; Moser, Claus; Wang, Heng-Zhuang; Høiby, Niels; Song, Zhi-Jun

    2015-01-01

    Formation of biofilm is a survival strategy for bacteria and fungi to adapt to their living environment, especially in the hostile environment. Under the protection of biofilm, microbial cells in biofilm become tolerant and resistant to antibiotics and the immune responses, which increases the difficulties for the clinical treatment of biofilm infections. Clinical and laboratory investigations demonstrated a perspicuous correlation between biofilm infection and medical foreign bodies or indwelling devices. Clinical observations and experimental studies indicated clearly that antibiotic treatment alone is in most cases insufficient to eradicate biofilm infections. Therefore, to effectively treat biofilm infections with currently available antibiotics and evaluate the outcomes become important and urgent for clinicians. The review summarizes the latest progress in treatment of clinical biofilm infections and scientific investigations, discusses the diagnosis and treatment of different biofilm infections and introduces the promising laboratory progress, which may contribute to prevention or cure of biofilm infections. We conclude that, an efficient treatment of biofilm infections needs a well-established multidisciplinary collaboration, which includes removal of the infected foreign bodies, selection of biofilm-active, sensitive and well-penetrating antibiotics, systemic or topical antibiotic administration in high dosage and combinations, and administration of anti-quorum sensing or biofilm dispersal agents. PMID:25504208

  14. Presence of exoY, exoS, exoU and exoT genes, antibiotic resistance and biofilm production among Pseudomonas aeruginosa isolates in Northwest Iran

    PubMed Central

    Azimi, Somayeh; Kafil, Hossein Samadi; Baghi, Hossein Bannazadeh; Shokrian, Saeed; Najaf, Khadijeh; Asgharzadeh, Mohammad; Yousefi, Mehdi; Shahrivar, Firooz; Aghazadeh, Mohammad

    2016-01-01

    Background: Pseudomonas aeruginosa, as Gram-negative rod bacilli, has an important role in human infection. In the present study we aimed to investigate the presence of exo genes and biofilm production among Pseudomonas aeruginosa isolates in Northwest Iran. Material and methods: 160 isolates of P. aeruginosa were collected and identified by biochemical tests and were characterized for antibiotic resistance. Biofilm production was evaluated by microtiter plate assay and the presence of exo genes was evaluated by allele-specific PCR (polymerase chain reaction). Chi-square test was used for statistical analysis. Results: The most effective antibiotics against isolates were colistin and polymyxin B. 87% of the isolates were biofilm producers of which 69% were strongly biofilm producers. 55% of the isolates carried exoY, 52% of the isolates carried exoU, and 26.3% and 5% carried exoS and exoT, respectively. Conclusion: Our findings showed different distribution of exo genes in clinical isolates of P. aeruginosa in Northwest Iran. ExoS and exoU were more prevalent in non-biofilm producers and exoY was more prevalent in biofilm producer isolates. These results might indicate the importance of exoY in biofilm production of Pseudomonas aeruginosa. PMID:26958458

  15. Presence of Pseudomonas aeruginosa influences biofilm formation and surface protein expression of Staphylococcus aureus.

    PubMed

    Kumar, Amit; Ting, Yen Peng

    2015-11-01

    Although Staphylococcus aureus and Pseudomonas aeruginosa can individually colonize and infect their hosts, the commensalistic effect of the two is more tenacious and lethal. In this study, it was shown that in co-culture with P. aeruginosa, a sub-population of S. aureus exhibited improved resistance to kanamycin by selection of small colony variant (SCV) phenotype. Additionally, biofilm formation by the two bacteria was denser in the co-culture, compared with biofilm formed in individual pure cultures. Using Atomic Force Microscope (AFM) force spectroscopy for single cells, it was demonstrated that S. aureus cultured in the presence of P. aeruginosa bound more tenaciously to substrates. Surface-shaved peptides were isolated and identified using ultra-performance liquid chromatography-quadrupole-time of flight and a homology search program spider. Results indicated that serine-rich adhesin, extracellular matrix binding protein and other putative adhesion proteins could be responsible for the enhanced attachment of S. aureus in the co-culture. Besides, several other proteins were differentially expressed, indicating the occurrence of a range of other interactions. Of particular interest was a multidrug resistant protein named ABC transporter permease which is known to expel xenobiotics out of the cells. Positive regulation of this protein could be involved in the SCV selection of S. aureus in the co-culture. PMID:25925222

  16. Comparative in vitro efficacies of various antipseudomonal antibiotics based catheter lock solutions on eradication of Pseudomonas aeruginosa biofilms.

    PubMed

    Ozbek, Berna; Mataraci-Kara, Emel

    2016-02-01

    Antibiotic lock technique (ALT) may be an adjunct therapy in treating catheter-related infections. The aim of this study was to determine the in vitro stability and efficacy of colistin, meropenem and levofloxacin alone or in combination with clarithromycin or heparin lock solutions against biofilm embedded Pseudomonas aeruginosa strains. The efficacy of antibiotic lock solutions was tested in an in vitro catheter biofilm model against P. aeruginosa isolated from catheter-related bacteremia. We observed that the use of meropenem, levofloxacin or colistin as a lock solution had potent bactericidal effects and could be prevented bacterial regrowth at 96 or 72 hours, respectively. When the tested antibiotics were used in combination with clarithromycin, the combinations were significantly more effective and rapid in eliminating P. aeruginosa colonization in biofilm than each of the antibiotics were used alone. Moreover, tested antibiotics in combination with heparin were not significantly different for killing effect against PA-1 and PA-27853 compared with that of each antibiotics alone. Tested catheter lock solutions may have promising adjuvants for treating infections caused by P. aeruginosa. PMID:25257204

  17. Regulatory and Metabolic Networks for the Adaptation of Pseudomonas aeruginosa Biofilms to Urinary Tract-Like Conditions

    PubMed Central

    Dohnt, Katrin; Haddad, Isam; Jänsch, Lothar; Klein, Johannes; Narten, Maike; Pommerenke, Claudia; Scheer, Maurice; Schobert, Max; Schomburg, Dietmar; Thielen, Bernhard; Jahn, Dieter

    2013-01-01

    Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections. PMID:23967252

  18. 2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

    PubMed

    Sethupathy, Sivasamy; Nithya, Chari; Pandian, Shunmugiah Karutha

    2015-01-01

    The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa. PMID:26571230

  19. Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

    PubMed

    Diaz De Rienzo, M A; Stevenson, P S; Marchant, R; Banat, I M

    2016-07-01

    Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds. PMID:26825819

  20. Flexible Survival Strategies of Pseudomonas aeruginosa in Biofilms Result in Increased Fitness Compared with Candida albicans *

    PubMed Central

    Purschke, Frauke Gina; Hiller, Ekkehard; Trick, Iris; Rupp, Steffen

    2012-01-01

    The majority of microorganisms persist in nature as surface-attached communities often surrounded by an extracellular matrix, called biofilms. Most natural biofilms are not formed by a single species but by multiple species. Microorganisms not only cooperate as in some multispecies biofilms but also compete for available nutrients. The Gram-negative bacterium Pseudomonas aeruginosa and the polymorphic fungus Candida albicans are two opportunistic pathogens that are often found coexisting in a human host. Several models of mixed biofilms have been reported for these organisms showing antagonistic behavior. To investigate the interaction of P. aeruginosa and C. albicans in more detail, we analyzed the secretome of single and mixed biofilms of both organisms using MALDI-TOF MS/MS at several time points. Overall 247 individual proteins were identified, 170 originated from P. aeruginosa and 77 from C. albicans. Only 39 of the 131 in mixed biofilms identified proteins were assigned to the fungus whereby the remaining 92 proteins belonged to P. aeruginosa. In single-species biofilms, both organisms showed a higher diversity of proteins with 73 being assigned to C. albicans and 154 to P. aeruginosa. Most interestingly, P. aeruginosa in the presence of C. albicans secreted 16 proteins in significantly higher amounts or exclusively among other virulence factors such as exotoxin A and iron acquisition systems. In addition, the high affinity iron-binding siderophore pyoverdine was identified in mixed biofilms but not in bacterial biofilms, indicating that P. aeruginosa increases its capability to sequester iron in competition with C. albicans. In contrast, C. albicans metabolism was significantly reduced, including a reduction in detectable iron acquisition proteins. The results obtained in this study show that microorganisms not only compete with the host for essential nutrients but also strongly with the present microflora in order to gain a competitive advantage. PMID

  1. Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

    PubMed

    Brown, A; Horobin, A; Blount, D G; Hill, P J; English, J; Rich, A; Williams, P M; Pritchard, D I

    2012-12-01

    In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata. PMID:22827809

  2. Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients.

    PubMed

    Ciofu, Oana; Tolker-Nielsen, Tim; Jensen, Peter Østrup; Wang, Hengzhuang; Høiby, Niels

    2015-05-01

    Lung infection is the main cause of morbidity and mortality in patients with cystic fibrosis and is mainly dominated by Pseudomonas aeruginosa. The biofilm mode of growth makes eradication of the infection impossible, and it causes a chronic inflammation in the airways. The general mechanisms of biofilm formation and antimicrobial tolerance and resistance are reviewed. Potential anti-biofilm therapeutic targets such as weakening of biofilms by quorum-sensing inhibitors or antibiotic killing guided by pharmacokinetics and pharmacodynamics of antibiotics are presented. The vicious circle of adaptive evolution of the persisting bacteria imposes important therapeutic challenges and requires development of new drug delivery systems able to reach the different niches occupied by the bacteria in the lung of cystic fibrosis patients. PMID:25477303

  3. Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection.

    PubMed

    Ibberson, Carolyn B; Parlet, Corey P; Kwiecinski, Jakub; Crosby, Heidi A; Meyerholz, David K; Horswill, Alexander R

    2016-06-01

    Staphylococcus aureus is a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureus biofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureus secretes HysA in order to cleave HA during infection. Through in vitro biofilm studies with HA, the hysA mutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysA expression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysA mutant biofilm infection compared to the S. aureus wild type. Histopathology demonstrated that infection with an hysA mutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureus HysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureus biofilm matrix and HysA is important for dissemination from a biofilm infection. PMID:27068096

  4. Lipopolysaccharide of Marinobacter litoralis inhibits swarming motility and biofilm formation in Pseudomonas aeruginosa PA01.

    PubMed

    Sardar, Raj Kumar; Kavita, Kumari; Jha, Bhavanath

    2015-06-01

    The lipopolysaccharide (LPS) was isolated from a marine bacterium identified as Marinobacter litoralis BK09 using 16S rRNA gene sequence similarity analysis. The GCMS analysis showed that the LPS contained 3-hydroxy-dodecanoic acid (C12:0 3OH) (49%), dodecanoic acid (C12:0) (24%) and decanoic acid (C10:0) (19%) as major fatty acids, and the polysaccharide constituents were fucose (53.79%), xylose (28.04%) and mannose (18.15%). The LPS almost completely inhibited swarming motility in Pseudomonas aeruginosa PA01. It also reduced biofilm formation by 50% with no adverse effect on cell growth. The production of virulence factor such as pyocyanin pigment was reduced (∼40%) by the LPS. The LPS did not show any limulus amoebocyte lysate (LAL) gelation activity. The repression of swarming motility, pyocyanin production and biofilm formation by the LPS suggests its potential application against P. aeruginosa infection. This is the first report on characterization and application of LPS from M. litoralis. PMID:25843881

  5. Studying the effect of alginate overproduction on Pseudomonas aeruginosa biofilm by atomic force microscopy.

    PubMed

    Lim, Jeesun; Cui, Yidan; Oh, Yoo Jin; Park, Ja Ryeong; Jo, William; Cho, You-Hee; Park, Sungsu

    2011-07-01

    Pseudomonas aeruginosa is the most important pathogen in cystic fibrosis patients and forms biofilms in the lung. P. aeruginosa strains isolated from the lungs of the patients have a mucoid phenotype overproducing alginate. The phenotype forms highly structured biofilms which are more resistant to antibiotics than biofilms formed by its nonmucoid phenotype. Conversion to the alginate-overproducing phenotype occurs through a mutation in rpoN gene in the strains. The biofilms formed by the alginate-overproducing phenotype are highly sticky, but their stickiness has not been measured. Herein, the stickiness of biofilms formed by the rpoN mutant was measured by atomic force microscopy (AFM). Confocal laser scanning microscopy showed that the biofilms formed by the slowly-growing rpoN mutant were more structured than those formed by the wild-type strain. AFM analysis indicated that the biofilms formed by the rpoN mutant were stickier than those formed by the wild type strain during the attachment and establishment stages, but the difference in stickiness was greatly reduced during the maturation stage possibly due to the cytosolic contents released from dead cells in the biofilms formed by the wild type. These results suggest that the alginate overproduction greatly affects the physical properties (topography and stickiness) of P. aeruginosa biofilms as well as the physiological properties (cell death and growth) of the bacterial cells inside the biofilms. PMID:22121590

  6. Effects of Chlorine Stress on Pseudomonas aeruginosa Biofilm and Analysis of Related Gene Expressions.

    PubMed

    Kekeç, Özge; Gökalsın, Barış; Karaltı, İskender; Kayhan, Figen Esin; Sesal, Nüzhet Cenk

    2016-08-01

    Chlorine is deployed worldwide to clean waters and prevent water-originated illnesses. However, chlorine has a limited disinfection capacity against biofilms. Microorganisms form biofilms to protect themselves from biological threats such as disinfectant chemicals. Pseudomonas aeruginosa is an opportunistic pathogen and its biofilm form attaches to surfaces, living buried into exopolysaccharides, can be present in all watery environments including tap water and drinking water. This research aimed to study the biofilm trigger mechanism of the opportunistic pathogen P. aeruginosa PAO1 strain, which is known to form biofilm in water supply systems and human body, under chlorine stress levels. In addition to biofilm staining, certain genes that are relevant to the stress condition were selected for gene expression analysis. The bacteria cultures were grown under chlorine stress with concentrations of 0.5, 0.7 and 1 mg/l. Six gene regions were determined related to biofilm and stress response: rpoS, bifA, migA, katB, soxR, and algC. Biofilm formation was analyzed by basic fuchsin staining, and gene expressions were quantified by quantitative real-time PCR. According to the results, highest biofilm production was observed in P. aeruginosa PAO1 wild strain under no stress conditions. Higher biofilm amounts were observed for bacteria under 0.5 and 0.7 mg/l chlorine stress compared to 1 mg/l chlorine stress. PMID:27146505

  7. Biofilm formation by clinical isolates and the implications in chronic infections

    PubMed Central

    2013-01-01

    Background Biofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR) species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics. Methods The biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC) from 2004–2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA) (n=23), Acinetobacter baumannii (n=53), Pseudomonas aeruginosa (n=36), Klebsiella pneumoniae (n=54), and Escherichia coli (n=39), were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM). Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro. Results Of the 205 clinical isolates tested, 126 strains (61.4%) were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro. Conclusions This

  8. Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines

    PubMed Central

    Okegbe, Chinweike; Harty, Colleen E.; Golub, Yuriy; Thao, Sandy; Ha, Dae Gon; Willger, Sven D.; O'Toole, George A.; Harwood, Caroline S.; Dietrich, Lars E. P.; Hogan, Deborah A.

    2014-01-01

    In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis. PMID:25340349

  9. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics

    PubMed Central

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E.; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the ‘holy grail’ in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  10. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

    PubMed Central

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  11. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa.

    PubMed

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  12. Disruption and eradication of P. aeruginosa biofilms using nitric oxide-releasing chitosan oligosaccharides

    PubMed Central

    Reighard, Katelyn P.; Hill, David B.; Dixon, Graham A.; Worley, Brittany; Schoenfisch, Mark H.

    2015-01-01

    Biofilm disruption and eradication were investigated as a function of nitric oxide- (NO) releasing chitosan oligosaccharide dose with results compared to control (ie non-NO-releasing) chitosan oligosaccharides and tobramycin. Quantification of biofilm expansion/contraction and multiple-particle tracking microrheology were used to assess the structural integrity of the biofilm before and after antibacterial treatment. While tobramycin had no effect on the physical properties of the biofilm, NO-releasing chitosan oligosaccharides exhibited dose-dependent behavior with biofilm degradation. Control chitosan oligosaccharides increased biofilm elasticity, indicating that the scaffold may mitigate the biofilm disrupting power of nitric oxide somewhat. The results from this study indicate that nitric oxide-releasing chitosan oligosaccharides act as dual-action therapeutics capable of eradicating and physically disrupting P. aeruginosa biofilms. PMID:26610146

  13. A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry.

    PubMed

    Park, Amber J; Murphy, Kathleen; Krieger, Jonathan R; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M

    2014-04-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  14. A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry*

    PubMed Central

    Park, Amber J.; Murphy, Kathleen; Krieger, Jonathan R.; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M.

    2014-01-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  15. Critical nitric oxide concentration for Pseudomonas aeruginosa biofilm reduction on polyurethane substrates.

    PubMed

    Neufeld, Bella H; Reynolds, Melissa M

    2016-01-01

    Bacterial colonies that reside on a surface, known as biofilms, are intrinsically impenetrable to traditional antibiotics, ultimately driving research toward an alternative therapeutic approach. Nitric oxide (NO) has gained attention for its biologically beneficial properties, particularly centered around its antibacterial capabilities. NO donors that can release the molecule under physiological conditions (such as S-nitrosothiols) can be utilized in clinical settings to combat bacterial biofilm infections. Herein the authors describe determining a critical concentration of NO necessary to cause >90% reduction of a Pseudomonas aeruginosa biofilm grown on medical grade polyurethane films. The biofilm was grown under optimal culture conditions [in nutrient broth media (NBM) at 37 °C] for 24 h before the addition of the NO donor S-nitrosoglutathione (GSNO) in NBM for an additional 24 h. The cellular viability of the biofilm after the challenge period was tested using varying concentrations of NO to determine the critical amount necessary to cause at least a 90% reduction in bacterial biofilm viability. The critical GSNO concentration was found to be 10 mM, which corresponds to 2.73 mM NO. Time kill experiments were performed on the 24 h biofilm using the critical amount of NO at 4, 8, 12, and 16 h and it was determined that the 90% biofilm viability reduction occurred at 12 h and was sustained for the entire 24 h challenge period. This critical concentration was subsequently tested for total NO release via a nitric oxide analyzer. The total amount of NO released over the 12 h challenge period was found to be 5.97 ± 0.66 × 10(-6) mol NO, which corresponds to 1.49 ± 0.17 μmol NO/ml NBM. This is the first identification of the critical NO concentration needed to elicit this biological response on a medically relevant polymer. PMID:27604080

  16. Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms.

    PubMed

    Pezzoni, Magdalena; Pizarro, Ramón A; Costa, Cristina S

    2014-02-01

    One of the more stressful factors that Pseudomonas aeruginosa must face in nature is solar UVA radiation. In this study, the protective role of KatA catalase in both planktonic cells and biofilms of P. aeruginosa against UVA radiation was determined by using the wild-type (PAO1) and an isogenic catalase deficient strain (katA). The katA strain was more sensitive than the wild-type, especially in the case of biofilms. Moreover, the wild-type biofilm was more resistant than its planktonic counterpart, but this was not observed in the katA strain. Striking KatA activity was detected in the matrix of katA(+) strains, and to our knowledge, this is the first report of this activity in the matrix of P. aeruginosa biofilms. Provision of bovine catalase or KatA to the matrix of a katA biofilm significantly increased its UVA tolerance, demonstrating that extracellular KatA is essential to optimal defense against UVA in P. aeruginosa biofilms. Efficiency of photocatalytic treatments using TiO2 and UVA was lower in biofilms than in planktonic cells, but KatA and KatB catalases seem not to be responsible for the higher resistance of the sessile cells to this treatment. PMID:24491420

  17. Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

    PubMed Central

    Purevdorj, B.; Costerton, J. W.; Stoodley, P.

    2002-01-01

    Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies. PMID:12200300

  18. A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

    PubMed

    Swearingen, Matthew C; Mehta, Ajeet; Mehta, Amar; Nistico, Laura; Hill, Preston J; Falzarano, Anthony R; Wozniak, Daniel J; Hall-Stoodley, Luanne; Stoodley, Paul

    2016-02-01

    Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples. PMID:26536894

  19. Delivery of tobramycin coupled to iron oxide nanoparticles across the biofilm of mucoidal Pseudonomas aeruginosa and investigation of its efficacy

    NASA Astrophysics Data System (ADS)

    Armijo, Leisha M.; Kopciuch, Michael; Olszá½¹wka, Zuzia; Wawrzyniec, Stephen J.; Rivera, Antonio C.; Plumley, John B.; Cook, Nathaniel C.; Brandt, Yekaterina I.; Huber, Dale L.; Smolyakov, Gennady A.; Adolphi, Natalie L.; Smyth, Hugh D. C.; Osiński, Marek

    2014-03-01

    Pseudomonas aeruginosa bacterium is a deadly pathogen, leading to respiratory failure in cystic fibrosis and nosocomial pneumonia, and responsible for high mortality rates in these diseases. P. aeruginosa has inherent as well as acquired resistance to many drug classes. In this paper, we investigate the effectiveness of two classes; aminoglycoside (tobramycin) and fluoroquinolone (ciprofloxacin) administered alone, as well as conjugated to iron oxide (magnetite) nanoparticles. P. aeruginosa possesses the ability to quickly alter its genetics to impart resistance to the presence of new, unrecognized treatments. As a response to this impending public health threat, we have synthesized and characterized magnetite nanoparticles capped with biodegradable short-chain carboxylic acid derivatives conjugated to common antibiotic drugs. The functionalized nanoparticles may carry the drug past the mucus and biofilm layers to target the bacterial colonies via magnetic gradient-guided transport. Additionally, the magnetic ferrofluid may be used under application of an oscillating magnetic field to raise the local temperature, causing biofilm disruption, slowed growth, and mechanical disruption. These abilities of the ferrofluid would also treat multi-drug resistant strains, which appear to be increasing in many nosocomial as well as acquired opportunistic infections. In this in vitro model, we show that the iron oxide alone can also inhibit bacterial growth and biofilm formation.

  20. 3-indolylacetonitrile decreases Escherichia coli O157:H7 biofilm formation and Pseudomonas aeruginosa virulence.

    PubMed

    Lee, Jin-Hyung; Cho, Moo Hwan; Lee, Jintae

    2011-01-01

    Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives are unstable in the microbial community because they are quickly degraded by diverse bacterial oxygenases. Hence, this work sought to identify novel, non-toxic, stable and potent indole derivatives from plant sources for inhibiting the biofilm formation of E. coli O157:H7 and P. aeruginosa. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit the biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN more effectively inhibited biofilms than indole for the two pathogenic bacteria. Additionally, IAN decreased the production of virulence factors including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), pyocyanin and pyoverdine in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, whereas IAN induced indole-related genes and prophage genes in E. coli O157:H7. It appeared that IAN inhibited the biofilm formation of E. coli by reducing curli formation and inducing indole production. Also, corroborating phenotypic results of P. aeruginosa, whole-transcriptomic data showed that IAN repressed virulence-related genes and motility-related genes, while IAN induced several small molecule transport genes. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa. Additionally, indole-3-carboxyaldehyde was another natural biofilm inhibitor for both E. coli and P. aeruginosa. PMID:20649646

  1. Loss of social behaviours in populations of Pseudomonas aeruginosa infecting lungs of patients with cystic fibrosis.

    PubMed

    Jiricny, Natalie; Molin, Søren; Foster, Kevin; Diggle, Stephen P; Scanlan, Pauline D; Ghoul, Melanie; Johansen, Helle Krogh; Santorelli, Lorenzo A; Popat, Roman; West, Stuart A; Griffin, Ashleigh S

    2014-01-01

    Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections. PMID:24454693

  2. Pseudomonas aeruginosa Biofilm Response and Resistance to Cold Atmospheric Pressure Plasma Is Linked to the Redox-Active Molecule Phenazine

    PubMed Central

    Mai-Prochnow, Anne; Bradbury, Mark; Ostrikov, Kostya; Murphy, Anthony B.

    2015-01-01

    Pseudomonas aeruginosa is an important opportunistic pathogen displaying high antibiotic resistance. Its resistance is in part due to its outstanding ability to form biofilms on a range of biotic and abiotic surfaces leading to difficult-to-treat, often long-term infections. Cold atmospheric plasma (CAP) is a new, promising antibacterial treatment to combat antibiotic-resistant bacteria. Plasma is ionized gas that has antibacterial properties through the generation of a mix of reactive oxygen and nitrogen species (RONS), excited molecules, charged particles and UV photons. Our results show the efficient removal of P. aeruginosa biofilms using a plasma jet (kINPen med), with no viable cells detected after 5 min treatment and no attached biofilm cells visible with confocal microscopy after 10 min plasma treatment. Because of its multi-factorial action, it is widely presumed that the development of bacterial resistance to plasma is unlikely. However, our results indicate that a short plasma treatment (3 min) may lead to the emergence of a small number of surviving cells exhibiting enhanced resistance to subsequent plasma exposure. Interestingly, these cells also exhibited a higher degree of resistance to hydrogen peroxide. Whole genome comparison between surviving cells and control cells revealed 10 distinct polymorphic regions, including four belonging to the redox active, antibiotic pigment phenazine. Subsequently, the interaction between phenazine production and CAP resistance was demonstrated in biofilms of transposon mutants disrupted in different phenazine pathway genes which exhibited significantly altered sensitivity to CAP. PMID:26114428

  3. Screening of Lactobacillus spp. for the prevention of Pseudomonas aeruginosa pulmonary infections

    PubMed Central

    2014-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host’s health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. Results Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. Conclusion Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further

  4. Nitroxides as anti-biofilm compounds for the treatment of Pseudomonas aeruginosa and mixed-culture biofilms.

    PubMed

    Alexander, Stefanie-Ann; Kyi, Caroline; Schiesser, Carl H

    2015-04-28

    A series of 23 nitroxides () was tested for biofilm modulatory activity using a crystal violet staining technique. 3-(Dodecane-1-thiyl)-4-(hydroxymethyl)-2,2,5,5-tetramethyl-1-pyrrolinoxyl () was found to significantly suppress biofilm formation and elicit dispersal events in both Pseudomonas aeruginosa and mixed-culture biofilms. Twitching and swarming motilities were enhanced by nitroxide , leaving the planktonic-specific swimming motility unaffected and suggesting that the mechanism of -mediated biofilm modulation is linked to the hyperactivation of surface-associated cell motilities. Preliminary structure-activity relationship studies identify the dodecanethiyl chain, hydroxymethyl substituent and the free radical moiety to be structural features pertinent to the anti-biofilm activity of . PMID:25804546

  5. MrkD1P from Klebsiella pneumoniae Strain IA565 Allows for Coexistence with Pseudomonas aeruginosa and Protection from Protease-Mediated Biofilm Detachment

    PubMed Central

    Childers, Brandon M.; Van Laar, Tricia A.; You, Tao; Clegg, Steven

    2013-01-01

    Biofilm formation and persistence are essential components for the continued survival of pathogens inside the host and constitute a major contributor to the development of chronic wounds with resistance to antimicrobial compounds. Understanding these processes is crucial for control of biofilm-mediated disease. Though chronic wound infections are often polymicrobial in nature, much of the research on chronic wound-related microbes has focused on single-species models. Klebsiella pneumoniae and Pseudomonas aeruginosa are microbes that are often found together in wound isolates and are able to form stable in vitro biofilms, despite the antagonistic nature of P. aeruginosa with other organisms. Mutants of the K. pneumoniae strain IA565 lacking the plasmid-borne mrkD1P gene were less competitive than the wild type in an in vitro dual-species biofilm model with P. aeruginosa (PAO1). PAO1 spent medium inhibited the formation of biofilm of mrkD1P-deficient mutants and disrupted preestablished biofilms, with no effect on IA565 and no effect on the growth of the wild type or mutants. A screen using a two-allele PAO1 transposon library identified the LasB elastase as the secreted effector involved in biofilm disruption, and a purified version of the protein produced results similar to those with PAO1 spent medium. Various other proteases had a similar effect, suggesting that the disruption of the mrkD1P gene causes sensitivity to general proteolytic effects and indicating a role for MrkD1P in protection against host antibiofilm effectors. Our results suggest that MrkD1P allows for competition of K. pneumoniae with P. aeruginosa in a mixed-species biofilm and provides defense against microbial and host-derived proteases. PMID:23980108

  6. An In vivo Wound Model Utilizing Bacteriophage Therapy of Pseudomonas aeruginosa Biofilms.

    PubMed

    Basu, Somprakas; Agarwal, Manav; Kumar Bhartiya, Satyanam; Nath, Gopal; Kumar Shukla, Vijay

    2015-08-01

    Bacteriophages have been used as effective therapy against bacterial biofilms on devices such as catheters, in the lungs such as in cystic fibrosis, and even in infected food. Unlike antibiotics, they are bacteria-specific and produce the desired effect without systemic complications; they can develop bacterial resistance, although in ways different from antibiotics. The present study aimed to assess the effect of bacteriophages against multidrug-resistant Pseudomonas aeruginosa in a mouse wound model. P. aeruginosa obtained from laboratory culture of burn wounds were characterized, harvested, and titrated, and biofilms were generated on sterile catheter sections (105 colony forming units/mL). Subcutaneous pockets were created on the backs of 24 male albino mice. Animals were randomized into 4 groups of 6 each. After evaluating a significant phage-bacteria interaction in vitro, 2 biofilm-laden catheter sections were implanted in subcutaneous pockets in mouse groups C and D. Sterile catheter sections only were implanted in group B. Group A had only a subcutaneous pocket without any catheter section. Phage cocktail solutions (10 \\'b5L of 107 phage forming units/mL) were injected daily in group D pockets only. Groups B and C received 10 \\'b5L of normal saline. After 10 days, the catheter sections were explanted from groups B, C, and D and tissue biopsy was taken from group A pockets and cultured for bacterial and phage colony counts. A significant drop in bacterial counts from 3.87 x 106 to 3.52 x 104 was observed in group D when compared with group C (3.87 x 106 to 3.85 x 105, P less than 0.05) A significant rise in the phage counts from 1 x 107 to 6.81 x 108 (P less than 0.05) also was observed in group D when compared with the baseline counts, indicating active phage proliferation and successful bacterial kill in group D. The present laboratory study could be indicative of a new treatment approach for multidrug-resistant bacterial infections, including wound

  7. Synergistic effect of membrane-active peptides polymyxin B and gramicidin S on multidrug-resistant strains and biofilms of Pseudomonas aeruginosa.

    PubMed

    Berditsch, Marina; Jäger, Thomas; Strempel, Nikola; Schwartz, Thomas; Overhage, Jörg; Ulrich, Anne S

    2015-09-01

    Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinical P. aeruginosa isolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa. PMID:26077259

  8. Community-based interference against integration of Pseudomonas aeruginosa into human salivary microbial biofilm.

    PubMed

    He, X; Hu, W; He, J; Guo, L; Lux, R; Shi, W

    2011-12-01

    As part of the human gastrointestinal tract, the oral cavity represents a complex biological system and harbors diverse bacterial species. Unlike the gut microbiota, which is often considered a health asset, studies of the oral commensal microbiota have been largely limited to their implication in oral conditions such as dental caries and periodontal disease. Less emphasis has been given to their potential beneficial roles, especially the protective effects against oral colonization by foreign or pathogenic bacteria. In this study, we used salivary microbiota derived from healthy human subjects to investigate protective effects against colonization and integration of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, into developing or pre-formed salivary biofilms. When co-cultivated in saliva medium, P. aeruginosa persisted in the planktonic phase, but failed to integrate into the salivary microbial community during biofilm formation. Furthermore, in saliva medium supplemented with sucrose, the oral microbiota inhibited the growth of P. aeruginosa by producing lactic acid. More interestingly, while pre-formed salivary biofilms were able to prevent P. aeruginosa colonization, the same biofilms recovered from mild chlorhexidine gluconate treatment displayed a shift in microbial composition and showed a drastic reduction in protection. Our study indicates that normal oral communities with balanced microbial compositions could be important in effectively preventing the integration of foreign or pathogenic bacterial species, such as P. aeruginosa. PMID:22053962

  9. Expeditive synthesis of trithiotriazine-cored glycoclusters and inhibition of Pseudomonas aeruginosa biofilm formation

    PubMed Central

    Smadhi, Meriem; Gingras, Marc; Abderrahim, Raoudha

    2014-01-01

    Summary Readily accessible, low-valency glycoclusters based on a triazine core bearing D-galactose and L-fucose epitopes are able to inhibit biofilm formation by Pseudomonas aeruginosa. These multivalent ligands are simple to synthesize, are highly soluble, and can be either homofunctional or heterofunctional. The galactose-decorated cluster shows good affinity for Pseudomonas aeruginosa lectin lecA. They are convenient biological probes for investigating the roles of lecA and lecB in biofilm formation. PMID:25246957

  10. Imaging Pseudomonas aeruginosa Biofilm Extracellular Polymer Scaffolds with Amphiphilic Carbon Dots.

    PubMed

    Ritenberg, Margarita; Nandi, Sukhendu; Kolusheva, Sofiya; Dandela, Rambabu; Meijler, Michael M; Jelinek, Raz

    2016-05-20

    Biofilm formation is a critical facet of pathogenesis and resilience of human, animal, and plant bacteria. Extracellular polymeric substances (EPS) constitute the physical scaffolding for bacterial biofilms and thus play central roles in their development and virulence. We show that newly synthesized amphiphilic fluorescent carbon dots (C-dots) readily bind to the EPS scaffold of Pseudomonas aeruginosa, a major biofilm-forming pathogen, resulting in unprecedented microscopic visualization of the EPS structural features. Fluorescence microscopy analysis utilizing the C-dots reveals that the P. aeruginosa EPS matrix exhibits a remarkable dendritic morphology. The experiments further illuminate the growth kinetics of the EPS and the effect of external factors such as temperature. We also show that the amphiphilic C-dot platform enabled screening of substances disrupting biofilm development, specifically quorum sensing inhibitors. PMID:26882175

  11. Development and (evidence for) destruction of biofilm with Pseudomonas aeruginosa as architect

    NASA Technical Reports Server (NTRS)

    Uzcategui, Valerie N.; Donadeo, John J.; Lombardi, Daniel R.; Costello, Michael J.; Sauer, Richard L.

    1991-01-01

    Disinfection and maintenance of an acceptable level of asepsis in spacecraft potable water delivery systems is a formidable task. The major area of research for this project has been to monitor the formation and growth of biofilm, and biofilm attached microorganisms, on stainless steel surfaces (specifically coupons), and the use of ozone for the elimination of these species in a closed loop system. A number of different techniques have been utilized during the course of a typical run. Scraping and sonication of coupon surfaces with subsequent plating as well as epifluorescence microscopy have been utilized to enumerate biofilm protected Pseudomonas aeruginosa. In addition, scanning electron microscopy is the method of choice to examine the integrity of the biofilm. For ozone determinations, the indigo decolorization spectrophotometric method seems most reliable. Both high- and low-nutrient cultured P. aeruginosa organisms were the target species for the ozone disinfection experiments.

  12. Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation.

    PubMed

    McElroy, Kerensa E; Hui, Janice G K; Woo, Jerry K K; Luk, Alison W S; Webb, Jeremy S; Kjelleberg, Staffan; Rice, Scott A; Thomas, Torsten

    2014-04-01

    Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10(-3) substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern short-term, within-population bacterial diversification. PMID:24706926

  13. Ciprofloxacin-Eluting Nanofibers Inhibits Biofilm Formation by Pseudomonas aeruginosa and a Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Ahire, Jayesh J.; Neveling, Deon P.; Hattingh, Melanie; Dicks, Leon M. T.

    2015-01-01

    Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with hospital-acquired infections and are known to form biofilms. Ciprofloxacin (CIP), which is normally used to treat these infections, is seldom effective in killing cells in a biofilm. This is mostly due to slow or weak penetration of CIP to the core of biofilms. The problem is accentuated by the release of CIP below MIC (minimal inhibitory concentration) levels following a rapid (burst) release. The aim of this study was to develop a drug carrier that would keep CIP above MIC levels for an extended period. Ciprofloxacin was suspended into poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO), and electrospun into nanofibers (CIP-F). All of the CIP was released from the nanofibers within 2 h, which is typical of a burst release. However, 99% of P. aeruginosa PA01 cells and 91% of S. aureus Xen 30 cells (a methicillin-resistant strain) in biofilms were killed when exposed to CIP-F. CIP levels remained above MIC for 5 days, as shown by growth inhibition of the cells in vitro. The nanofibers were smooth in texture with no bead formation, as revealed by scanning electron and atomic force microscopy. A single vibration peak at 1632 cm-1, recorded with Fourier transform infrared spectroscopy, indicated that CIP remained in crystal form when incorporated into PDLLA: PEO. No abnormalities in the histology of MCF-12A breast epithelial cells were observed when exposed to CIP-F. This is the first report of the inhibition of biofilm formation by CIP released from PDLLA: PEO nanofibers. PMID:25853255

  14. The gallium(III)-salicylidene acylhydrazide complex shows synergistic anti-biofilm effect and inhibits toxin production by Pseudomonas aeruginosa.

    PubMed

    Rzhepishevska, Olena; Hakobyan, Shoghik; Ekstrand-Hammarström, Barbro; Nygren, Yvonne; Karlsson, Torbjörn; Bucht, Anders; Elofsson, Mikael; Boily, Jean-François; Ramstedt, Madeleine

    2014-09-01

    Bacterial biofilms cause a range of problems in many areas and especially in health care. Biofilms are difficult to eradicate with traditional antibiotics and consequently there is a need for alternative ways to prevent and/or remove bacterial biofilms. Furthermore, the emergence of antibiotic resistance in bacteria creates a challenge to find new types of antibiotics with a lower evolutionary pressure for resistance development. One route to develop such drugs is to target the so called virulence factors, i.e. bacterial systems used when bacteria infect a host cell. This study investigates synergy effects between Ga(III) ions, previously reported to suppress biofilm formation and growth in bacteria, and salicylidene acylhydrazides (hydrazones) that have been proposed as antivirulence drugs targeting the type three secretion system used by several Gram-negative pathogens, including Pseudomonas aerugionosa, during bacterial infection of host cells. A library of hydrazones was screened for: Fe(III) binding, enhanced anti-biofilm effect with Ga(III) on P. aeruginosa, and low cytotoxicity to mammalian cells. The metal coordination for the most promising ligand, 2-Oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (ME0163) with Ga(III) was investigated using extended X-ray absorption fine structure spectroscopy as well as density functional theory. The results showed that Ga(III) chelates the hydrazone with 5- and 6-membered chelating rings, and that the Ga(III)-ME0163 complex enhanced the antibiofilm effect of Ga(III) while suppressing the type three secretion system in P. aeruginosa. The latter effect was not observed for the hydrazone alone and was similar for Ga(III)-citrate and Ga(III)-ME0163 complexes, indicating that the inhibition of virulence was caused by Ga(III). PMID:24837331

  15. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin

    PubMed Central

    Tseng, Boo Shan; Zhang, Wei; Harrison, Joe J.; Quach, Tam P.; Song, Jisun Lee; Penterman, Jon; Singh, Pradeep K.; Chopp, David L.; Packman, Aaron I.; Parsek, Matthew R.

    2013-01-01

    SUMMARY Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the biofilm matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance, as previous studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid P. aeruginosa biofilms. To our surprise, we showed that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions, but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration. PMID:23751003

  16. The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Billings, Nicole; Ramirez Millan, Maria; Caldara, Marina; Rusconi, Roberto; Tarasova, Yekaterina; Stocker, Roman; Ribbeck, Katharina

    2013-01-01

    Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development. PMID:23950711

  17. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections

    PubMed Central

    Winstanley, Craig; O’Brien, Siobhan; Brockhurst, Michael A.

    2016-01-01

    Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification. PMID:26946977

  18. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections.

    PubMed

    Winstanley, Craig; O'Brien, Siobhan; Brockhurst, Michael A

    2016-05-01

    Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification. PMID:26946977

  19. Plasma-Mediated Inactivation of Pseudomonas aeruginosa Biofilms Grown on Borosilicate Surfaces under Continuous Culture System

    PubMed Central

    Vandervoort, Kurt G.; Brelles-Mariño, Graciela

    2014-01-01

    Biofilms are microbial communities attached to a surface and embedded in a matrix composed of exopolysaccharides and excreted nucleic acids. Bacterial biofilms are responsible for undesirable effects such as disease, prostheses colonization, biofouling, equipment damage, and pipe plugging. Biofilms are also more resilient than free-living cells to regular sterilization methods and therefore it is indispensable to develop better ways to control and remove them. The use of gas discharge plasmas is a good alternative since plasmas contain a mixture of reactive agents well-known for their decontamination potential against free microorganisms. We have previously reported that Pseudomonas aeruginosa biofilms were inactivated after a 1-min plasma exposure. We determined that the adhesiveness and the thickness of Pseudomonas biofilms grown on borosilicate were reduced. We also reported sequential morphological changes and loss of viability upon plasma treatment. However, the studies were carried out in batch cultures. The use of a continuous culture results in a more homogenous environment ensuring reproducible biofilm growth. The aim of this work was to study plasma-mediated inactivation of P. aeruginosa biofilms grown on borosilicate in a continuous culture system. In this paper we show that biofilms grown on glass under continuous culture can be inactivated by using gas discharge plasma. Both biofilm architecture and cell culturabilty are impacted by the plasma treatment. The inactivation kinetics is similar to previously described ones and cells go through sequential changes ranging from minimal modification without loss of viability at short plasma exposure times, to major structure and viability loss at longer exposure times. We report that changes in biofilm structure leading to the loss of culturability and viability are related to a decrease of the biofilm matrix adhesiveness. To our knowledge, there has been no attempt to evaluate the inactivation

  20. The BioFilm Ring Test: a Rapid Method for Routine Analysis of Pseudomonas aeruginosa Biofilm Formation Kinetics

    PubMed Central

    Olivares, Elodie; Badel-Berchoux, Stéphanie; Provot, Christian; Jaulhac, Benoît; Prévost, Gilles; Bernardi, Thierry

    2015-01-01

    Currently, few techniques are available for the evaluation of bacterial biofilm adhesion. These detection tools generally require time for culture and/or arduous handling steps. In this work, the BioFilm Ring Test (BRT), a new technology, was used to estimate the biofilm formation kinetics of 25 strains of Pseudomonas aeruginosa, isolated from the sputum of cystic fibrosis (CF) patients. The principle of the new assay is based on the mobility measurement of magnetic microbeads mixed with a bacterial suspension in a polystyrene microplate. If free to move under the magnetic action, particles gather to a visible central spot in the well bottom. Therefore, the absence of spot formation in the plate reflects the bead immobilization by a biofilm in formation. The BRT device allowed us to classify the bacterial strains into three general adhesion profiles. Group 1 consists of bacteria, which are able to form a solid biofilm in <2 h. Group 2 comprises the strains that progressively set up a biofilm during 24 h. Lastly, group 3 includes the strains that stay in a planktonic form. The grouping of our strains did not differ according to culture conditions, i.e., the use of different sets of beads or culture media. The BRT is shown to be an informative tool for the characterization of biofilm-forming bacteria. Various application perspectives may be investigated for this device, such as the addition of antibiotics to the bacterial suspension to select which would have the ability to inhibit the biofilm formation. PMID:26719437

  1. The BioFilm Ring Test: a Rapid Method for Routine Analysis of Pseudomonas aeruginosa Biofilm Formation Kinetics.

    PubMed

    Olivares, Elodie; Badel-Berchoux, Stéphanie; Provot, Christian; Jaulhac, Benoît; Prévost, Gilles; Bernardi, Thierry; Jehl, François

    2016-03-01

    Currently, few techniques are available for the evaluation of bacterial biofilm adhesion. These detection tools generally require time for culture and/or arduous handling steps. In this work, the BioFilm Ring Test (BRT), a new technology, was used to estimate the biofilm formation kinetics of 25 strains of Pseudomonas aeruginosa, isolated from the sputum of cystic fibrosis (CF) patients. The principle of the new assay is based on the mobility measurement of magnetic microbeads mixed with a bacterial suspension in a polystyrene microplate. If free to move under the magnetic action, particles gather to a visible central spot in the well bottom. Therefore, the absence of spot formation in the plate reflects the bead immobilization by a biofilm in formation. The BRT device allowed us to classify the bacterial strains into three general adhesion profiles. Group 1 consists of bacteria, which are able to form a solid biofilm in <2 h. Group 2 comprises the strains that progressively set up a biofilm during 24 h. Lastly, group 3 includes the strains that stay in a planktonic form. The grouping of our strains did not differ according to culture conditions, i.e., the use of different sets of beads or culture media. The BRT is shown to be an informative tool for the characterization of biofilm-forming bacteria. Various application perspectives may be investigated for this device, such as the addition of antibiotics to the bacterial suspension to select which would have the ability to inhibit the biofilm formation. PMID:26719437

  2. Pseudomonas aeruginosa Exhibits Deficient Biofilm Formation in the Absence of Class II and III Ribonucleotide Reductases Due to Hindered Anaerobic Growth

    PubMed Central

    Crespo, Anna; Pedraz, Lucas; Astola, Josep; Torrents, Eduard

    2016-01-01

    Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this

  3. Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

    PubMed Central

    Baker, Perrin; Hill, Preston J.; Snarr, Brendan D.; Alnabelseya, Noor; Pestrak, Matthew J.; Lee, Mark J.; Jennings, Laura K.; Tam, John; Melnyk, Roman A.; Parsek, Matthew R.; Sheppard, Donald C.; Wozniak, Daniel J.; Howell, P. Lynne

    2016-01-01

    Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics. PMID:27386527

  4. Evaluation of antibiotic effects on Pseudomonas aeruginosa biofilm using Raman spectroscopy and multivariate analysis

    PubMed Central

    Jung, Gyeong Bok; Nam, Seong Won; Choi, Samjin; Lee, Gi-Ja; Park, Hun-Kuk

    2014-01-01

    We investigate the mode of action and classification of antibiotic agents (ceftazidime, patulin, and epigallocatechin gallate; EGCG) on Pseudomonas aeruginosa (P. aeruginosa) biofilm using Raman spectroscopy with multivariate analysis, including support vector machine (SVM) and principal component analysis (PCA). This method allows for quantitative, label-free, non-invasive and rapid monitoring of biochemical changes in complex biofilm matrices with high sensitivity and specificity. In this study, the biofilms were grown and treated with various agents in the microfluidic device, and then transferred onto gold-coated substrates for Raman measurement. Here, we show changes in biochemical properties, and this technology can be used to distinguish between changes induced in P. aeruginosa biofilms using three antibiotic agents. The Raman band intensities associated with DNA and proteins were decreased, compared to control biofilms, when the biofilms were treated with antibiotics. Unlike with exposure to ceftazidime and patulin, the Raman spectrum of biofilms exposed to EGCG showed a shift in the spectral position of the CH deformation stretch band from 1313 cm−1 to 1333 cm−1, and there was no difference in the band intensity at 1530 cm−1 (C = C stretching, carotenoids). The PCA-SVM analysis results show that antibiotic-treated biofilms can be detected with high sensitivity of 93.33%, a specificity of 100% and an accuracy of 98.33%. This method also discriminated the three antibiotic agents based on the cellular biochemical and structural changes induced by antibiotics with high sensitivity and specificity of 100%. This study suggests that Raman spectroscopy with PCA-SVM is potentially useful for the rapid identification and classification of clinically-relevant antibiotics of bacteria biofilm. Furthermore, this method could be a powerful approach for the development and screening of new antibiotics. PMID:25401035

  5. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays.

    PubMed

    Holguín, Angela V; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C; Barrios, Andrés Fernando González; Vives, Martha J

    2015-08-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

  6. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

    PubMed Central

    Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

    2015-01-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

  7. Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Franklin, Michael J.; Williamson, Kerry S.; Folsom, James P.; Boegli, Laura; James, Garth A.

    2015-01-01

    Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin. PMID:25870065

  8. Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.

    PubMed

    Stewart, Philip S; Franklin, Michael J; Williamson, Kerry S; Folsom, James P; Boegli, Laura; James, Garth A

    2015-07-01

    Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin. PMID:25870065

  9. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.

    PubMed

    Tseng, Boo Shan; Zhang, Wei; Harrison, Joe J; Quach, Tam P; Song, Jisun Lee; Penterman, Jon; Singh, Pradeep K; Chopp, David L; Packman, Aaron I; Parsek, Matthew R

    2013-10-01

    Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid Pseudomonas aeruginosa biofilms. To our surprise, we found that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration. PMID:23751003

  10. The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

    PubMed Central

    Moscoso, Joana A.; Jaeger, Tina; Valentini, Martina; Hui, Kailyn; Jenal, Urs

    2014-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism. PMID:25225264

  11. Biofilms in Infections of the Eye

    PubMed Central

    Bispo, Paulo J. M.; Haas, Wolfgang; Gilmore, Michael S.

    2015-01-01

    The ability to form biofilms in a variety of environments is a common trait of bacteria, and may represent one of the earliest defenses against predation. Biofilms are multicellular communities usually held together by a polymeric matrix, ranging from capsular material to cell lysate. In a structure that imposes diffusion limits, environmental microgradients arise to which individual bacteria adapt their physiologies, resulting in the gamut of physiological diversity. Additionally, the proximity of cells within the biofilm creates the opportunity for coordinated behaviors through cell–cell communication using diffusible signals, the most well documented being quorum sensing. Biofilms form on abiotic or biotic surfaces, and because of that are associated with a large proportion of human infections. Biofilm formation imposes a limitation on the uses and design of ocular devices, such as intraocular lenses, posterior contact lenses, scleral buckles, conjunctival plugs, lacrimal intubation devices and orbital implants. In the absence of abiotic materials, biofilms have been observed on the capsule, and in the corneal stroma. As the evidence for the involvement of microbial biofilms in many ocular infections has become compelling, developing new strategies to prevent their formation or to eradicate them at the site of infection, has become a priority. PMID:25806622

  12. Catheter-related infections caused by Pseudomonas aeruginosa: virulence factors involved and their relationships.

    PubMed

    Olejnickova, Katerina; Hola, Veronika; Ruzicka, Filip

    2014-11-01

    The nosocomial pathogen Pseudomonas aeruginosa is equipped with a large arsenal of cell-associated and secreted virulence factors which enhance its invasive potential. The complex relationships among virulence determinants have hitherto not been fully elucidated. In the present study, 175 catheter-related isolates were observed for the presence of selected virulence factors, namely extracellular enzymes and siderophore production, biofilm formation, resistance to antibiotics, and motility. A high percentage of the strains produced most of the tested virulence factors. A positive correlation was identified between the production of several exoproducts, and also between the formation of both types of biofilm. An opposite trend was observed between the two types of biofilm and the production of siderophores. Whereas the relationship between the submerged biofilm production (i.e. the biofilm formed on the solid surface below the water level) and the siderophore secretion was negative, the production of air-liquid interface (A-L) biofilm (i.e. the biofilm floating on the surface of the cultivation medium) and the siderophore secretion were positively correlated. All correlations were statistically significant at the level P = 0.05 with the correlation coefficient γ ≥ 0.50. Our results suggest that: (1) the co-production of the lytic enzymes and siderophores can play an important role in the pathogenesis of the catheter-related infections and should be taken into account when the virulence potential is assessed; (2) biofilm-positive strains are capable of forming both submerged and non-attached A-L biofilms; and (3) the different micro-environment in the submerged biofilm and A-L biofilm layers have opposite consequences for the production of other virulence factors. PMID:24842562

  13. Metabolomics-Based Screening of Biofilm-Inhibitory Compounds against Pseudomonas aeruginosa from Burdock Leaf.

    PubMed

    Lou, Zaixiang; Tang, Yuxia; Song, Xinyi; Wang, Hongxin

    2015-01-01

    Screening of anti-biofilm compounds from the burdock leaf based on metabolomics is reported here. The crystal violet assay indicated 34% ethanol elution fraction of burdock leaf could completely inhibit biofilm formation of Pseudomonas aeruginosa at 1 mg·mL(-1). Then, the chemical composition of burdock leaf fraction was analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and 11 active compounds (chlorogenic acid, caffeic acid, p-coumaric acid, quercetin, ursolic acid, rutin, cynarin, luteolin, crocin, benzoic acid, and Tenacissoside I) were identified. Lastly, UPLC-MS analysis was employed to obtain the metabolic fingerprints of burdock leaf fractions before and after inhibiting the biofilm of Pseudomonas aeruginosa. The metabolic fingerprints were transformed to data, analyzed with PLS-DA (partial least squares discriminant analysis) and the peaks whose area was significantly changed were found out. Thus, 81 compounds were screened as potential anti-biofilm ingredients. Among them, rutin, ursolic acid, caffeic acid, p-coumaric acid and quercetin were identified and confirmed as the main anti-biofilm compounds in burdock leaf. The study provided basic anti-biofilm profile data for the compounds in burdock leaf, as well as provided a convenient method for fast screening of anti-biofilm compounds from natural plants. PMID:26370951

  14. Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics

    PubMed Central

    Zhang, Li; Fritsch, Meredith; Hammond, Lisa; Landreville, Ryan; Slatculescu, Cristina; Colavita, Antonio; Mah, Thien-Fah

    2013-01-01

    Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of P. aeruginosa UCBPP-PA14 (ndvB, PA1875–1877 and tssC1) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents. PMID:23637868

  15. Microbiologically Influenced Corrosion of 2707 Hyper-Duplex Stainless Steel by Marine Pseudomonas aeruginosa Biofilm.

    PubMed

    Li, Huabing; Zhou, Enze; Zhang, Dawei; Xu, Dake; Xia, Jin; Yang, Chunguang; Feng, Hao; Jiang, Zhouhua; Li, Xiaogang; Gu, Tingyue; Yang, Ke

    2016-01-01

    Microbiologically Influenced Corrosion (MIC) is a serious problem in many industries because it causes huge economic losses. Due to its excellent resistance to chemical corrosion, 2707 hyper duplex stainless steel (2707 HDSS) has been used in the marine environment. However, its resistance to MIC was not experimentally proven. In this study, the MIC behavior of 2707 HDSS caused by the marine aerobe Pseudomonas aeruginosa was investigated. Electrochemical analyses demonstrated a positive shift in the corrosion potential and an increase in the corrosion current density in the presence of the P. aeruginosa biofilm in the 2216E medium. X-ray photoelectron spectroscopy (XPS) analysis results showed a decrease in Cr content on the coupon surface beneath the biofilm. The pit imaging analysis showed that the P. aeruginosa biofilm caused a largest pit depth of 0.69 μm in 14 days of incubation. Although this was quite small, it indicated that 2707 HDSS was not completely immune to MIC by the P. aeruginosa biofilm. PMID:26846970

  16. Microbiologically Influenced Corrosion of 2707 Hyper-Duplex Stainless Steel by Marine Pseudomonas aeruginosa Biofilm

    NASA Astrophysics Data System (ADS)

    Li, Huabing; Zhou, Enze; Zhang, Dawei; Xu, Dake; Xia, Jin; Yang, Chunguang; Feng, Hao; Jiang, Zhouhua; Li, Xiaogang; Gu, Tingyue; Yang, Ke

    2016-02-01

    Microbiologically Influenced Corrosion (MIC) is a serious problem in many industries because it causes huge economic losses. Due to its excellent resistance to chemical corrosion, 2707 hyper duplex stainless steel (2707 HDSS) has been used in the marine environment. However, its resistance to MIC was not experimentally proven. In this study, the MIC behavior of 2707 HDSS caused by the marine aerobe Pseudomonas aeruginosa was investigated. Electrochemical analyses demonstrated a positive shift in the corrosion potential and an increase in the corrosion current density in the presence of the P. aeruginosa biofilm in the 2216E medium. X-ray photoelectron spectroscopy (XPS) analysis results showed a decrease in Cr content on the coupon surface beneath the biofilm. The pit imaging analysis showed that the P. aeruginosa biofilm caused a largest pit depth of 0.69 μm in 14 days of incubation. Although this was quite small, it indicated that 2707 HDSS was not completely immune to MIC by the P. aeruginosa biofilm.

  17. Microbiologically Influenced Corrosion of 2707 Hyper-Duplex Stainless Steel by Marine Pseudomonas aeruginosa Biofilm

    PubMed Central

    Li, Huabing; Zhou, Enze; Zhang, Dawei; Xu, Dake; Xia, Jin; Yang, Chunguang; Feng, Hao; Jiang, Zhouhua; Li, Xiaogang; Gu, Tingyue; Yang, Ke

    2016-01-01

    Microbiologically Influenced Corrosion (MIC) is a serious problem in many industries because it causes huge economic losses. Due to its excellent resistance to chemical corrosion, 2707 hyper duplex stainless steel (2707 HDSS) has been used in the marine environment. However, its resistance to MIC was not experimentally proven. In this study, the MIC behavior of 2707 HDSS caused by the marine aerobe Pseudomonas aeruginosa was investigated. Electrochemical analyses demonstrated a positive shift in the corrosion potential and an increase in the corrosion current density in the presence of the P. aeruginosa biofilm in the 2216E medium. X-ray photoelectron spectroscopy (XPS) analysis results showed a decrease in Cr content on the coupon surface beneath the biofilm. The pit imaging analysis showed that the P. aeruginosa biofilm caused a largest pit depth of 0.69 μm in 14 days of incubation. Although this was quite small, it indicated that 2707 HDSS was not completely immune to MIC by the P. aeruginosa biofilm. PMID:26846970

  18. Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis

    PubMed Central

    Shirazi, Fazal; Ferreira, Jose A. G.; Stevens, David A.; Clemons, Karl V.; Kontoyiannis, Dimitrios P.

    2016-01-01

    Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation. PMID:26930399

  19. The inhibition of Pseudomonas aeruginosa biofilm formation by micafungin and the enhancement of antimicrobial agent effectiveness in BALB/c mice.

    PubMed

    Kissoyan, Kohar Annie B; Bazzi, Wael; Hadi, Usamah; Matar, Ghassan M

    2016-08-01

    Micafungin inhibits biofilm formation by impeding 1,3-β-D-glucan synthesis in Candida albicans. Since Pseudomonas aeruginosa also has 1,3-β-D-glucan in its cell wall, this study assessed the effects of antibacterial agents in vitro and in vivo on micafungin-treated biofilm-forming P. aeruginosa isolates. After treatment with micafungin as well as with a panel of four antibacterial agents, biofilm production was significantly reduced as measured by spectrophotometry. The relative mRNA transcription levels for the genes encoding pellicles (pelC) and cell wall 1,3-β-D-glucan (ndvB), which were measured by quantitative reverse transcription PCR (qRT-PCR), significantly decreased with micafungin treatment. In vivo, the survival rates of P. aeruginosa-infected BALB/c mice significantly increased after combined treatment with micafungin and each of the antibacterial agents. Of these treatments, the combination of micafungin with levofloxacin had the highest survival rate; this combination was the most effective treatment against P. aeruginosa-induced infection. PMID:27347641

  20. Glucose Starvation-Induced Dispersal of Pseudomonas aeruginosa Biofilms Is cAMP and Energy Dependent

    PubMed Central

    Huynh, Tran T.; McDougald, Diane; Klebensberger, Janosch; Al Qarni, Budoor; Barraud, Nicolas; Rice, Scott A.; Kjelleberg, Staffan; Schleheck, David

    2012-01-01

    Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival. PMID:22905180

  1. Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Plyuta, Vladimir; Zaitseva, Julia; Lobakova, Elena; Zagoskina, Natalia; Kuznetsov, Alexander; Khmel, Inessa

    2013-11-01

    In the natural environment, bacteria predominantly exist in matrix-enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3- to 7-fold under the action of vanillin and epicatechin, and 2- to 2.5-fold in the presence of 4-hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4-hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N-3-oxo-dodecanoyl-homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N-acyl-homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs. PMID:23594262

  2. A peptide from human β thymosin as a platform for the development of new anti-biofilm agents for Staphylococcus spp. and Pseudomonas aeruginosa.

    PubMed

    Schillaci, Domenico; Spinello, Angelo; Cusimano, Maria Grazia; Cascioferro, Stella; Barone, Giampaolo; Vitale, Maria; Arizza, Vincenzo

    2016-08-01

    Conventional antibiotics might fail in the treatment of biofilm-associated infections causing infection recurrence and chronicity. The search for antimicrobial peptides has been performed with the aim to discover novel anti-infective agents active on pathogens in both planktonic and biofilm associated forms. The fragment 9-19 of human thymosin β4 was studied through 1 μs MD simulation. Two main conformations of the peptide were detected, both constituted by a central hydrophobic core and by the presence of peripheral charged residues suggesting a possible mechanism of interaction with two models of biological membranes, related to eukaryotic or bacterial membrane respectively. In addition, the peptide was chemically synthesized and its antimicrobial activity was tested in vitro against planktonic and biofilm form of a group of reference strains of Staphylococcus spp. and one P. aeruginosa strain. The human thymosin β4 fragment EIEKFDKSKLK showed antibacterial activity against staphylococcal strains and Pseudomonas aeruginosa ATCC 15442 at concentrations from 12.5 to 6.2 mg/ml and inhibited biofilm formation at sub-inhibitory concentrations (3.1-0.75 mg/ml). The activity of the fragment in inhibiting biofilm formation, could be due to the conformations highlighted by the MD simulations, suggesting its interaction with the bacterial membrane. Human thymosin β4 fragment can be considered a promising lead compound to develop novel synthetic or recombinant derivatives with improved pharmaceutical potential. PMID:27339305

  3. 6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition

    PubMed Central

    Kim, Han-Shin; Lee, Sang-Hoon; Byun, Youngjoo; Park, Hee-Deung

    2015-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors via quorum sensing (QS). Interfering with normal QS interactions between signal molecules and their cognate receptors is a developing strategy for attenuating its virulence. Here we tested the hypothesis that 6-gingerol, a pungent oil of fresh ginger, reduces biofilm formation and virulence by antagonistically binding to P. aeruginosa QS receptors. In silico studies demonstrated molecular binding occurs between 6-gingerol and the QS receptor LasR through hydrogen bonding and hydrophobic interactions. Experimentally 6-gingerol reduced biofilm formation, several virulence factors (e.g., exoprotease, rhamnolipid, and pyocyanin), and mice mortality. Further transcriptome analyses demonstrated that 6-gingerol successfully repressed QS-induced genes, specifically those related to the production of virulence factors. These results strongly support our hypothesis and offer insight into the molecular mechanism that caused QS gene repression. PMID:25728862

  4. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  5. In vitro and in vivo effects of sub-MICs of pexiganan and imipenem on Pseudomonas aeruginosa adhesion and biofilm development.

    PubMed

    Cirioni, Oscar; Silvestri, Carmela; Ghiselli, Roberto; Kamysz, Wojciech; Minardi, Daniele; Castelli, Pamela; Orlando, Fiorenza; Kamysz, Elzbieta; Provinciali, Mauro; Muzzonigro, Giovanni; Guerrieri, Mario; Giacometti, Andrea

    2013-12-01

    An in vitro and in vivo study was performed to quantify adhesion and biofilm formation ability of Pseudomonas aeruginosa slime producer under the effect of sub-minimal inhibitory concentrations (MICs) of pexiganan and imipenem. To evaluate adherence, squares of ureteral stents were placed in six-well tissue-culture plates containing 6 ml of a cell suspension grown in the presence of sub-MICs of study antibiotics. To evaluate biofilm formation sterilized squares were placed in six-well tissue culture plates containing 6 ml of triptic soy broth (TSB) supplemented with 0.25% of glucose and the respective amount of antibiotic. For in vivo study a biofilm infection rat model was performed. The study included an uninfected control group to evaluate the sterility of surgical procedure, a group infected with a slime-producer P. aeruginosa strain not previously treated with antibiotics and two groups infected with the strain previously treated with imipenem or pexiganan. Adherence and biofilm in vitro formation was strongly affected by pre-treatment with pexiganan and imipenem, with the latter being the more effective antibiotic. The in vivo results showed a reduction in bacterial load on the ureteral stent tissue of the pre-treated strain. Differently, urine cultures showed no differences in bacterial growth for the pre-treated strain showing that it retained its ability to cause infection. This study suggests that sub-MIC imipenem and pexiganan could be a good strategy to target the adhesion process during the infection cycle. PMID:24335459

  6. Interactions of Methicillin Resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in Polymicrobial Wound Infection

    PubMed Central

    Pastar, Irena; Nusbaum, Aron G.; Gil, Joel; Patel, Shailee B.; Chen, Juan; Valdes, Jose; Stojadinovic, Olivera; Plano, Lisa R.; Tomic-Canic, Marjana; Davis, Stephen C.

    2013-01-01

    Understanding the pathology resulting from Staphylococcus aureus and Pseudomonas aeruginosa polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant S. aureus USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and P. aeruginosa on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo. Polymicrobial wound infection in the presence of P. aeruginosa resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections. PMID:23451098

  7. Interactions of Pseudomonas aeruginosa in predominant biofilm or planktonic forms of existence in mixed culture with Escherichia coli in vitro.

    PubMed

    Kuznetsova, Marina V; Maslennikova, Irina L; Karpunina, Tamara I; Nesterova, Larisa Yu; Demakov, Vitaly A

    2013-09-01

    Pseudomonas aeruginosa and Escherichia coli are known to be involved in mixed communities in diverse niches. In this study we examined the influence of the predominant form of cell existence of and the exometabolite production by P. aeruginosa strains on interspecies interactions, in vitro. Bacterial numbers of P. aeruginosa and E. coli in mixed plankton cultures and biofilms compared with their numbers in single plankton cultures and biofilms changed in a different way, but were in accordance with the form of P. aeruginosa cell existence. The mass of a mixed-species biofilm was greater than the mass of a single-species biofilm. Among the mixed biofilms, the one with the "planktonic" P. aeruginosa strain had the least biomass. The total pyocyanin and pyoverdin levels were found to be lower in all mixed plankton cultures. Despite this, clinical P. aeruginosa strains irrespective of the predominant form of existence ("biofilm" or "planktonic") had a higher total concentration of exometabolites than did the reference strain in 12-24 h mixed cultures. The metabolism of E. coli, according to its bioluminescence, was reduced in mixed cultures, and the decrease was by 20- to 100-fold greater with the clinical Pseudomonas strains than the reference Pseudomonas strain. Thus, both the predominant form of existence of and the exometabolite production by distinct P. aeruginosa strains should be considered to fully understand the interspecies relationship and bacteria survival in natural communities. PMID:24011343

  8. Disrupting the mixed-species biofilm of Klebsiella pneumoniae B5055 and Pseudomonas aeruginosa PAO using bacteriophages alone or in combination with xylitol.

    PubMed

    Chhibber, Sanjay; Bansal, Shruti; Kaur, Sukhmandir

    2015-07-01

    We investigated the potential of bacteriophages alone as well as in combination with xylitol for tackling mixed-species biofilm of Pseudomonas aeruginosa and Klebsiella pneumoniae. When mixed-species biofilm was established on polycarbonate discs, P. aeruginosa formed the base layer which was physically shielded on the top by K. pneumoniae. Thereafter, mixed-species biofilm was treated with bacteriophages. K. pneumoniae-specific depolymerase-producing phage KPO1K2 caused significant reduction in the count of Klebsiella. In contrast, P. aeruginosa-specific non-depolymerase-producing phage Pa29 failed to cause any reduction in the count of Pseudomonas. However, application of both phages together resulted in significant reduction in the count of both organisms. This suggests that depolymerase produced by phage KPO1K2 hydrolysed the top layer of K. pneumoniae and guided the entry of Pa29 to reach P. aeruginosa lying underneath. This phenomenon was confirmed when K. pneumoniae-specific non-depolymerase-producing phage NDP was used along with Pa29. Pa29 could not penetrate and reach its host bacterium. Xylitol worked synergistically along with the phage, resulting in a significant decrease in counts of both organisms. Disruption of mixed species biofilm by phage and xylitol was confirmed on the basis of the amount of protein and DNA released. This phage-based approach to altering the structural pattern and disrupting the mixed species biofilm is the first of its kind. It can be used as a topical application, a coating for foreign bodies or for aerosol delivery to tackle infections where both pathogens coexist in a biofilm mode. PMID:25922418

  9. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material.

    PubMed Central

    Nickel, J C; Ruseska, I; Wright, J B; Costerton, J W

    1985-01-01

    When disks of urinary catheter material were exposed to the flow of artificial urine containing cells of Pseudomonas aeruginosa, a thick adherent biofilm, composed of these bacteria and of their exopolysaccharide products, developed on the latex surface within 8 h. After this colonization, sterile artificial urine containing 1,000 micrograms of tobramycin per ml was flowed past this established biofilm, and a significant proportion of the bacterial cells within the biofilm were found to be still viable after 12 h of exposure to this very high concentration of aminoglycoside antibiotic. Planktonic (floating) cells taken from the test system just before the exposure of the biofilm to the antibiotic were completely killed by 50 micrograms of tobramycin per ml. The MIC of tobramycin for cells taken from the seeding cultures before colonization of the catheter material, and for surviving cells recovered directly from the tobramycin-treated biofilm, was found to be 0.4 micrograms/ml when dispersed cells were assayed by standard methods. These data indicate that growth within thick adherent biofilms confers a measure of tobramycin resistance on cells of P. aeruginosa. Images PMID:3923925

  10. Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis

    PubMed Central

    2010-01-01

    Background Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared ranki ngs for a priori identified physiological marker genes between the biofilm and published data sets. Results Biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database http://www.ncbi.nlm.nih.gov/geo. By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h-1. Conclusions Collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity

  11. Mep72, a Metzincin Protease That Is Preferentially Secreted by Biofilms of Pseudomonas aeruginosa

    PubMed Central

    Passmore, Ian J.; Nishikawa, Kahoko; Lilley, Kathryn S.; Bowden, Steven D.; Chung, Jade C. S.

    2014-01-01

    In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors. PMID:25488299

  12. Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa.

    PubMed

    Passmore, Ian J; Nishikawa, Kahoko; Lilley, Kathryn S; Bowden, Steven D; Chung, Jade C S; Welch, Martin

    2015-02-15

    In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors. PMID:25488299

  13. Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms.

    PubMed

    Grobe, K J; Zahller, J; Stewart, P S

    2002-07-01

    Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case, resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration. PMID:12080421

  14. c-di-GMP and its Effects on Biofilm Formation and Dispersion: a Pseudomonas Aeruginosa Review.

    PubMed

    Ha, Dae-Gon; O'Toole, George A

    2015-04-01

    Since its initial discovery as an allosteric factor regulating cellulose biosynthesis in Gluconacetobacter xylinus, the list of functional outputs regulated by c-di-GMP has grown. We have focused this article on one of these c-di-GMP-regulated processes, namely, biofilm formation in the organism Pseudomonas aeruginosa. The majority of diguanylate cyclases and phosphodiesterases encoded in the P. aeruginosa genome still remain uncharacterized; thus, there is still a great deal to be learned about the link between c-di-GMP and biofilm formation in this microbe. In particular, while a number of c-di-GMP metabolizing enzymes have been identified that participate in reversible and irreversible attachment and biofilm maturation, there is a still a significant knowledge gap regarding the c-di-GMP output systems in this organism. Even for the well-characterized Pel system, where c-di-GMP-mediated transcriptional regulation is now well documented, how binding of c-di-GMP by PelD stimulates Pel production is not understood in any detail. Similarly, c-di-GMP-mediated control of swimming, swarming and twitching also remains to be elucidated. Thus, despite terrific advances in our understanding of P. aeruginosa biofilm formation and the role of c-di-GMP in this process since the last version of this book (indeed there was no chapter on c-di-GMP!) there is still much to learn. PMID:26104694

  15. Unique Biofilm Signature, Drug Susceptibility and Decreased Virulence in Drosophila through the Pseudomonas aeruginosa Two-Component System PprAB

    PubMed Central

    Giraud, Caroline; Bernard, Christophe S.; Calderon, Virginie; Ewald, Friederike; Plésiat, Patrick; Nguyen, Cathy; Grunwald, Didier; Attree, Ina; Jeannot, Katy; Fauvarque, Marie-Odile

    2012-01-01

    Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections. PMID:23209420

  16. Impact of growth temperature and surface type on the resistance of Pseudomonas aeruginosa and Staphylococcus aureus biofilms to disinfectants.

    PubMed

    Abdallah, Marwan; Khelissa, Oussama; Ibrahim, Ali; Benoliel, Corinne; Heliot, Laurent; Dhulster, Pascal; Chihib, Nour-Eddine

    2015-12-01

    Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus on food-contact-surfaces represents a significant risk for the public health. In this context, the present study investigates the relationship between the environmental conditions of biofilm formation and the resistance to disinfectants. Therefore, a static biofilm reactor, called NEC-Biofilm System, was established in order to study the effect of growth temperature (20, 30 and 37°C), and of the surface type (stainless steel and polycarbonate), on biofilm resistance to disinfectants. These conditions were selected to mimic the biofilm formation on abiotic surfaces of food processing industries. The antibiofilm assays were performed on biofilms grown during 24 h. The results showed that the growth temperature influenced significantly the biofilm resistance to disinfectants. These data also revealed that the growth temperature has a significant effect on the biofilm structure of both bacteria. Furthermore, the increase of the biofilm growth temperature increased significantly the algD transcript level in sessile P. aeruginosa cells, whereas the icaA one was not affected in S. aureus cells. Overall, our findings show that the biofilm structure and matrix cannot fully explain the biofilm resistance to disinfectant agents. Nevertheless, it underlines the intimate link between environmental conditions, commonly met in food sectors, and the biofilm resistance to disinfectants. PMID:26233298

  17. Deciphering the Contribution of Biofilm to the Pathogenesis of Peritoneal Dialysis Infections: Characterization and Microbial Behaviour on Dialysis Fluids

    PubMed Central

    Sampaio, Joana; Machado, Diana; Gomes, Ana Marta; Machado, Idalina; Santos, Cledir; Lima, Nelson; Carvalho, Maria João; Cabrita, António

    2016-01-01

    Infections are major complications in peritoneal dialysis (PD) with a multifactorial etiology that comprises patient, microbial and dialytic factors. This study aimed at investigating the contribution of microbial biofilms on PD catheters to recalcitrant infections and their interplay with PD related-factors. A prospective observational study was performed on 47 patients attending Centro Hospitalar of Porto and Vila Nova de Gaia/Espinho to whom the catheter was removed due to infectious (n = 16) and non-infectious causes (n = 31). Microbial density on the catheter was assessed by culture methods and the isolated microorganisms identified by matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry. The effect of conventional and three biocompatible PD solutions on 16 Coagulase Negative Staphylococci (CNS) and 10 Pseudomonas aeruginosa strains planktonic growth and biofilm formation was evaluated. Cultures were positive in 87.5% of the catheters removed due infectious and 90.3% removed due to non-infectious causes. However, microbial yields were higher on the cuffs of catheters removed due to infection vs. non-infection. Staphylococci (CNS and Staphylococcus aureus) and P. aeruginosa were the predominant species: 32% and 20% in the infection and 43.3% and 22.7% in the non-infection group, respectively. In general, PD solutions had a detrimental effect on planktonic CNS and P. aeruginosa strains growth. All strains formed biofilms in the presence of PD solutions. The solutions had a more detrimental effect on P. aeruginosa than CNS strains. No major differences were observed between conventional and biocompatible solutions, although in icodextrin solution biofilm biomass was lower than in bicarbonate/lactate solution. Overall, we show that microbial biofilm is universal in PD catheters with the subclinical menace of Staphylococci and P. aeruginosa. Cuffs colonization may significantly contribute to infection. PD solutions differentially

  18. Coordination of Swarming Motility, Biosurfactant Synthesis, and Biofilm Matrix Exopolysaccharide Production in Pseudomonas aeruginosa

    PubMed Central

    Wang, Shiwei; Yu, Shan; Zhang, Zhenyin; Wei, Qing; Yan, Lu; Ai, Guomin; Liu, Hongsheng

    2014-01-01

    Biofilm formation is a complex process in which many factors are involved. Bacterial swarming motility and exopolysaccharides both contribute to biofilm formation, yet it is unclear how bacteria coordinate swarming motility and exopolysaccharide production. Psl and Pel are two key biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen has three types of motility, swimming, twitching, and swarming. In this study, we found that elevated Psl and/or Pel production reduced the swarming motility of P. aeruginosa but had little effect on swimming and twitching. The reduction was due to decreased rhamnolipid production with no relation to the transcription of rhlAB, two key genes involved in the biosynthesis of rhamnolipids. Rhamnolipid-negative rhlR and rhlAB mutants synthesized more Psl, whereas exopolysaccharide-deficient strains exhibited a hyperswarming phenotype. These results suggest that competition for common sugar precursors catalyzed by AlgC could be a tactic for P. aeruginosa to balance the synthesis of exopolysaccharides and rhamnolipids and to control bacterial motility and biofilm formation inversely because the biosynthesis of rhamnolipids, Psl, and Pel requires AlgC to provide the sugar precursors and an additional algC gene enhances the biosynthesis of Psl and rhamnolipids. In addition, our data indicate that the increase in RhlI/RhlR expression attenuated Psl production. This implied that the quorum-sensing signals could regulate exopolysaccharide biosynthesis indirectly in bacterial communities. In summary, this study represents a mechanism that bacteria utilize to coordinate swarming motility, biosurfactant synthesis, and biofilm matrix exopolysaccharide production, which is critical for biofilm formation and bacterial survival in the environment. PMID:25172852

  19. Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide

    PubMed Central

    Elkins, James G.; Hassett, Daniel J.; Stewart, Philip S.; Schweizer, Herbert P.; McDermott, Timothy R.

    1999-01-01

    The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H2O2) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H2O2. Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking the H2O2-inducible catalase KatB, was similar to the wild-type strain with respect to H2O2 resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H2O2, while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H2O2. Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H2O2, suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H2O2, while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of β-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H2O2, particularly at high H2O2

  20. Predictive Computer Models for Biofilm Detachment Properties in Pseudomonas aeruginosa.

    PubMed

    Cogan, Nick G; Harro, Janette M; Stoodley, Paul; Shirtliff, Mark E

    2016-01-01

    Microbial biofilm communities are protected against environmental extremes or clearance by antimicrobial agents or the host immune response. They also serve as a site from which microbial populations search for new niches by dispersion via single planktonic cells or by detachment by protected biofilm aggregates that, until recently, were thought to become single cells ready for attachment. Mathematically modeling these events has provided investigators with testable hypotheses for further study. Such was the case in the recent article by Kragh et al. (K. N. Kragh, J. B. Hutchison, G. Melaugh, C. Rodesney, A. E. Roberts, Y. Irie, P. Ø. Jensen, S. P. Diggle, R. J. Allen, V. Gordon, and T. Bjarnsholt, mBio 7:e00237-16, 2016, http://dx.doi.org/10.1128/mBio.00237-16), in which investigators were able to identify the differential competitive advantage of biofilm aggregates to directly attach to surfaces compared to the single-celled planktonic populations. Therefore, as we delve deeper into the properties of the biofilm mode of growth, not only do we need to understand the complexity of biofilms, but we must also account for the properties of the dispersed and detached populations and their effect on reseeding. PMID:27302761

  1. Predictive Computer Models for Biofilm Detachment Properties in Pseudomonas aeruginosa

    PubMed Central

    Cogan, Nick G.; Harro, Janette M.; Stoodley, Paul

    2016-01-01

    ABSTRACT Microbial biofilm communities are protected against environmental extremes or clearance by antimicrobial agents or the host immune response. They also serve as a site from which microbial populations search for new niches by dispersion via single planktonic cells or by detachment by protected biofilm aggregates that, until recently, were thought to become single cells ready for attachment. Mathematically modeling these events has provided investigators with testable hypotheses for further study. Such was the case in the recent article by Kragh et al. (K. N. Kragh, J. B. Hutchison, G. Melaugh, C. Rodesney, A. E. Roberts, Y. Irie, P. Ø. Jensen, S. P. Diggle, R. J. Allen, V. Gordon, and T. Bjarnsholt, mBio 7:e00237-16, 2016, http://dx.doi.org/10.1128/mBio.00237-16), in which investigators were able to identify the differential competitive advantage of biofilm aggregates to directly attach to surfaces compared to the single-celled planktonic populations. Therefore, as we delve deeper into the properties of the biofilm mode of growth, not only do we need to understand the complexity of biofilms, but we must also account for the properties of the dispersed and detached populations and their effect on reseeding. PMID:27302761

  2. Characterization of membrane lipidome changes in Pseudomonas aeruginosa during biofilm growth on glass wool.

    PubMed

    Benamara, Hayette; Rihouey, Christophe; Abbes, Imen; Ben Mlouka, Mohamed Amine; Hardouin, Julie; Jouenne, Thierry; Alexandre, Stéphane

    2014-01-01

    Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria. PMID:25265483

  3. Characterization of Membrane Lipidome Changes in Pseudomonas aeruginosa during Biofilm Growth on Glass Wool

    PubMed Central

    Benamara, Hayette; Rihouey, Christophe; Abbes, Imen; Ben Mlouka, Mohamed Amine; Hardouin, Julie; Jouenne, Thierry; Alexandre, Stéphane

    2014-01-01

    Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria. PMID:25265483

  4. Inhibitory effect of biocides on the viable masses and matrices of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

    PubMed

    Toté, K; Horemans, T; Vanden Berghe, D; Maes, L; Cos, P

    2010-05-01

    Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides. PMID:20363795

  5. Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

    PubMed Central

    Facchini, Marcella; De Fino, Ida; Riva, Camilla; Bragonzi, Alessandra

    2014-01-01

    A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host. This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease. This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies. PMID:24686327

  6. Development of biofilm-targeted antimicrobial wound dressing for the treatment of chronic wound infections.

    PubMed

    Ng, Shiow-Fern; Leow, Hon-Lunn

    2015-01-01

    It has been established that microbial biofilms are largely responsible for the recalcitrance of many wound infections to conventional antibiotics. It was proposed that the efficacy of antibiotics could be optimized via the inhibition of bacterial biofilm growth in wounds. The combination of antibiofilm agent and antibiotics into a wound dressing may be a plausible strategy in wound infection management. Xylitol is an antibiofilm agent that has been shown to inhibit the biofilm formation. The purpose of this study was to develop an alginate film containing xylitol and gentamicin for the treatment of wound infection. Three films, i.e. blank alginate film (SA), alginate film with xylitol (F5) and alginate film with xylitol and gentamicin (AG), were prepared. The films were studied for their physical properties, swelling ratio, moisture absorption, moisture vapor transmission rate (MVTR), mechanical and rheology properties, drug content uniformity as well as in vitro drug release properties. Antimicrobial and antibiofilm in vitro studies on Staphylococcus aureus and Pseudomonas aeruginosa were also performed. The results showed that AG demonstrates superior mechanical properties, rheological properties and a higher MVTR compared with SA and F5. The drug flux of AG was higher than that of commercial gentamicin cream. Furthermore, antimicrobial studies showed that AG is effective against both S. aureus and P. aeruginosa, and the antibiofilm assays demonstrated that the combination was effective against biofilm bacteria. In summary, alginate films containing xylitol and gentamicin may potentially be used as new dressings for the treatment of wound infection. PMID:25758412

  7. Characterization of Pseudomonas aeruginosa isolated from chronically infected children with cystic fibrosis in India

    PubMed Central

    Agarwal, Gunjan; Kapil, Arti; Kabra, Susheel Kumar; Das, Bimal Kumar; Dwivedi, Sada Nand

    2005-01-01

    Background Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods. Results We studied 56 patients with CF attending the Pediatric Chest clinic at All India Institute of Medical Sciences, New Delhi, India during August 1998-August 2001. These patients were regularly followed up at the clinic. Out of 56 patients, 27 were culture positive for P. aeruginosa where 8 were chronically infected (Group1) and 19 were intermittently colonized with the organism (Group2). Patients under Group1 had significantly higher rates of hospitalization, death and colonization with different colony morphotypes (p < 0.05). The isolates from Group1 patients were the positive producers of extended spectrum beta lactamase. A total of 5 auxotrophs were recovered from 2 patients where one was chronically infected with P. aeruginosa and the other was a recently enrolled patient. The auxotrophs had the specific requirement for methionine and arginine. Molecular typing revealed 33 ERIC-PCR (E1-E33) and 5 PCR-ribotyping (P1-P5) patterns. By ERIC-PCR, 4 patients were colonized with 2–4 genotypes and the remaining 23 patients were colonized with the single genotype. Conclusion With chronicity of infection, P. aeruginosa becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and shows auxotrophy for amino acids. The chronically infected patients can be colonized with multiple genotypes. Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF

  8. The chemical digestion of Ti6Al7Nb scaffolds produced by Selective Laser Melting reduces significantly ability of Pseudomonas aeruginosa to form biofilm.

    PubMed

    Junka, Adam F; Szymczyk, Patrycja; Secewicz, Anna; Pawlak, Andrzej; Smutnicka, Danuta; Ziółkowski, Grzegorz; Bartoszewicz, Marzenna; Chlebus, Edward

    2016-01-01

    In our previous work we reported the impact of hydrofluoric and nitric acid used for chemical polishing of Ti-6Al-7Nb scaffolds on decrease of the number of Staphylococcus aureus biofilm forming cells. Herein, we tested impact of the aforementioned substances on biofilm of Gram-negative microorganism, Pseudomonas aeruginosa, dangerous pathogen responsible for plethora of implant-related infections. The Ti-6Al-7Nb scaffolds were manufactured using Selective Laser Melting method. Scaffolds were subjected to chemical polishing using a mixture of nitric acid and fluoride or left intact (control group). Pseudomonal biofilm was allowed to form on scaffolds for 24 hours and was removed by mechanical vortex shaking. The number of pseudomonal cells was estimated by means of quantitative culture and Scanning Electron Microscopy. The presence of nitric acid and fluoride on scaffold surfaces was assessed by means of IR and rentgen spetorscopy. Quantitative data were analysed using the Mann-Whitney test (P ≤ 0.05). Our results indicate that application of chemical polishing correlates with significant drop of biofilm-forming pseudomonal cells on the manufactured Ti-6Al-7Nb scaffolds ( p = 0.0133, Mann-Whitney test) compared to the number of biofilm-forming cells on non-polished scaffolds. As X-ray photoelectron spectroscopy revealed the presence of fluoride and nitrogen on the surface of scaffold, we speculate that drop of biofilm forming cells may be caused by biofilm-supressing activity of these two elements. PMID:27150429

  9. Delayed Wound Healing in Diabetic (db/db) Mice with Pseudomonas aeruginosa Biofilm Challenge – A Model for the Study of Chronic Wounds

    PubMed Central

    Zhao, Ge; Hochwalt, Phillip C.; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.

    2010-01-01

    Chronic wounds are a major clinical problem that leads to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models. The goal of this study was to create a reproducible chronic wound model in diabetic mice by application of bacterial biofilm. Six millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms two days post-wounding, and covered with semi-occlusive dressings for two weeks. Most of the control wounds were epithelialized by 28 days post-wounding. In contrast, none of biofilm challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis and epidermal hyperplasia adjacent to challenged wounds- all indicators of an inflammatory non-healing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality and demonstrated delayed wound healing following biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing. PMID:20731798

  10. Evaluation of adhesive and anti-adhesive properties of Pseudomonas aeruginosa biofilms and their inhibition by herbal plants

    PubMed Central

    Zameer, Farhan; MS, Rukmangada; Chauhan, Jyoti Bala; Khanum, Shaukath Ara; Kumar, Pramod; Devi, Aishwarya Tripurasundari; MN, Nagendra Prasad; BL, Dhananjaya

    2016-01-01

    Background and Objectives: Adhesion and colonization are prerequisites for the establishment of bacterial pathogenesis. The biofilm development of Pseudomonas aeruginosa was assessed on adhesive surfaces like dialysis membrane, stainless steel, glass and polystyrene. Materials and Methods: Microtiter plate biofilm assay was performed to assess the effect of nutrient medium and growth parameters of P. aeruginosa. Further, its growth on adhesive surfaces namely hydrophilic (dialysis membrane) and hydrophobic (polystyrene plate, square glass and stainless steel coupon) was assessed. The exopolysaccharide (EPS) was quantified using ruthenium red microplate assay and microscopic analysis was used to observe P. aeruginosa biofilm architecture. The anti-biofilm activity of herbal extracts on mature P. aeruginosa was performed. Results: The formation of large scale biofilms on dialysis membrane for 72 h was proved to be the best surface. In microscopic studies, very few exopolysaccaride fibrils, indicating a rather loose matrix was observed at 48 h. Further, thick exopolysaccaride, indicated higher adhesive properties at 72 h which is evident from ruthenium red staining. Among the plant extract used, Justicia wynaadensis leaf and Aristolochia indica (Eswari) root extract showed significant reduction of anti-biofilm activity of 0.178 OD and 0.192 OD in inhibiting mature biofilms at 0.225 OD respectively, suggesting the possible use of these extracts as efficient anti-adhesive and biofilm-disrupting agents with potential applications in controlling biofilms on surfaces. Conclusion: Our study facilitates better understanding in the development of P. aeruginosa biofilms on different food processing and clinical surfaces ultimately taking care of food safety and hygiene. PMID:27307976

  11. The diguanylate cyclase GcbA facilitates Pseudomonas aeruginosa biofilm dispersion by activating BdlA.

    PubMed

    Petrova, Olga E; Cherny, Kathryn E; Sauer, Karin

    2015-01-01

    Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion. PMID:25331436

  12. The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

    PubMed Central

    Petrova, Olga E.; Cherny, Kathryn E.

    2014-01-01

    Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion. PMID:25331436

  13. A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

    PubMed Central

    Bijtenhoorn, Patrick; Mayerhofer, Hubert; Müller-Dieckmann, Jochen; Utpatel, Christian; Schipper, Christina; Hornung, Claudia; Szesny, Matthias; Grond, Stephanie; Thürmer, Andrea; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Dierking, Katja; Schulenburg, Hinrich; Streit, Wolfgang R.

    2011-01-01

    In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies. PMID:22046268

  14. Influence of chelation strength and bacterial uptake of gallium salicylidene acylhydrazide on biofilm formation and virulence of Pseudomonas aeruginosa.

    PubMed

    Hakobyan, Shoghik; Rzhepishevska, Olena; Björn, Erik; Boily, Jean-François; Ramstedt, Madeleine

    2016-07-01

    Development of antibiotic resistance in bacteria causes major challenges for our society and has prompted a great need for new and alternative treatment methods for infection. One promising approach is to target bacterial virulence using for example salicylidene acylhydrazides (hydrazones). Hydrazones coordinate metal ions such as Fe(III) and Ga(III) through a five-membered and a six-membered chelation ring. One suggested mode of action is via restricting bacterial Fe uptake. Thus, it was hypothesized that the chelating strength of these substances could be used to predict their biological activity on bacterial cells. This was investigated by comparing Ga chelation strength of two hydrazone complexes, as well as bacterial Ga uptake, biofilm formation, and virulence in the form of production and secretion of a toxin (ExoS) by Pseudomonas aeruginosa. Equilibrium constants for deprotonation and Ga(III) binding of the hydrazone N'-(5-chloro-2-hydroxy-3-methylbenzylidene)-2,4-dihydroxybenzhydrazide (ME0329), with anti-virulence effect against P. aeruginosa, were determined and compared to bacterial siderophores and the previously described Ga(III) 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (Ga-ME0163) and Ga-citrate complexes. In comparison with these two complexes, it was shown that the uptake of Ga(III) was higher from the Ga-ME0329 complex. The results further show that the Ga-ME0329 complex reduced ExoS expression and secretion to a higher extent than Ga-citrate, Ga-ME0163 or the non-coordinated hydrazone. However, the effect against biofilm formation by P. aeruginosa, by the ME0329 complex, was similar to Ga-citrate and lower than what has been reported for Ga-ME0163. PMID:27118030

  15. In Vitro Antibiofilm Efficacies of Different Antibiotic Combinations with Zinc Sulfate against Pseudomonas aeruginosa Recovered from Hospitalized Patients with Urinary Tract Infection

    PubMed Central

    Elkhatib, Walid; Noreddin, Ayman

    2014-01-01

    Urinary tract infections (UTIs) are a serious healthcare dilemma influencing millions of patients every year and represent the second most frequent type of body infection. Pseudomonas aeruginosa is a multidrug-resistant pathogen causing numerous chronic biofilm-associated infections including urinary tract, nosocomial, and medical devices-related infections. In the present study, the biofilm of P. aeruginosa CCIN34519, recovered from inpatients with UTIs, was established on polystyrene substratum and scanning electron microscopy (SEM) and was utilized for visualization of the biofilm. A previously described in vitro system for real-time monitoring of biofilm growth/inhibition was utilized to assess the antimicrobial effects of ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, ertapenem, ceftriaxone, gentamicin, and tobramycin as single antibiotics as well as in combinations with zinc sulfate (2.5 mM) against P. aeruginosa CCIN34519 biofilm. Meanwhile, minimum inhibitory concentrations (MICs) at 24 h and mutant prevention concentrations (MPCs) at 96 h were determined for the aforementioned antibiotics. The real-time monitoring data revealed diverse responses of P. aeruginosa CCIN34519 biofilm to the tested antibiotic-zinc sulfate combinations with potential synergisms in cases of fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin, and norfloxacin) and carbapenem (ertapenem) as demonstrated by reduced MIC and MPC values. Conversely, considerable antagonisms were observed with cephalosporin (ceftriaxone) and aminoglycosides (gentamicin, and tobramycin) as shown by substantially increased MICs and MPCs values. Further deliberate in vivo investigations for the promising synergisms are required to evaluate their therapeutic potentials for treatment of UTIs caused by P. aeruginosa biofilms as well as for developing preventive strategies. PMID:27025734

  16. Efflux as a glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms.

    PubMed

    Vikram, Amit; Bomberger, Jennifer M; Bibby, Kyle J

    2015-01-01

    A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. PMID:25824217

  17. Efflux as a Glutaraldehyde Resistance Mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa Biofilms

    PubMed Central

    Vikram, Amit; Bomberger, Jennifer M.

    2015-01-01

    A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. PMID:25824217

  18. Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs

    PubMed Central

    Malone, Jacob G

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that predominates during the later stages of cystic fibrosis (CF) lung infections. Over many years of chronic lung colonization, P. aeruginosa undergoes extensive adaptation to the lung environment, evolving both toward a persistent, low virulence state and simultaneously diversifying to produce a number of phenotypically distinct morphs. These lung-adapted P. aeruginosa strains include the small colony variants (SCVs), small, autoaggregative isolates that show enhanced biofilm formation, strong attachment to surfaces, and increased production of exopolysaccharides. Their appearance in the sputum of CF patients correlates with increased resistance to antibiotics, poor lung function, and prolonged persistence of infection, increasing their relevance as a subject for clinical investigation. The evolution of SCVs in the CF lung is associated with overproduction of the ubiquitous bacterial signaling molecule cyclic-di-GMP, with increased cyclic-di-GMP levels shown to be responsible for the SCV phenotype in a number of different CF lung isolates. Here, we review the current state of research in clinical P. aeruginosa SCVs. We will discuss the phenotypic characteristics underpinning the SCV morphotype, the clinical implications of lung colonization with SCVs, and the molecular basis and clinical evolution of the SCV phenotype in the CF lung environment. PMID:26251621

  19. Cell-to-cell signaling and Pseudomonas aeruginosa infections.

    PubMed Central

    Van Delden, C.; Iglewski, B. H.

    1998-01-01

    Pseudomonas aeruginosa is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium's virulence depends on a large number of cell-associated and extracellular factors. Cell-to-cell signaling systems control the expression and allow a coordinated, cell-density-dependent production of many extracellular virulence factors. We discuss the possible role of cell-to-cell signaling in the pathogenesis of P. aeruginosa infections and present a rationale for targeting cell-to-cell signaling systems in the development of new therapeutic approaches. PMID:9866731

  20. Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa

    PubMed Central

    Pewzner-Jung, Yael; Tavakoli Tabazavareh, Shaghayegh; Grassmé, Heike; Becker, Katrin Anne; Japtok, Lukasz; Steinmann, Jörg; Joseph, Tammar; Lang, Stephan; Tuemmler, Burkhard; Schuchman, Edward H; Lentsch, Alex B; Kleuser, Burkhard; Edwards, Michael J; Futerman, Anthony H; Gulbins, Erich

    2014-01-01

    Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection. PMID:25085879

  1. Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections

    PubMed Central

    Cigana, Cristina; Lorè, Nicola Ivan; Riva, Camilla; De Fino, Ida; Spagnuolo, Lorenza; Sipione, Barbara; Rossi, Giacomo; Nonis, Alessandro; Cabrini, Giulio; Bragonzi, Alessandra

    2016-01-01

    Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies. PMID:26883959

  2. Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections.

    PubMed

    Cigana, Cristina; Lorè, Nicola Ivan; Riva, Camilla; De Fino, Ida; Spagnuolo, Lorenza; Sipione, Barbara; Rossi, Giacomo; Nonis, Alessandro; Cabrini, Giulio; Bragonzi, Alessandra

    2016-01-01

    Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies. PMID:26883959

  3. BswR controls bacterial motility and biofilm formation in Pseudomonas aeruginosa through modulation of the small RNA rsmZ

    PubMed Central

    Wang, Chao; Ye, Fuzhou; Kumar, Veerendra; Gao, Yong-Gui; Zhang, Lian-Hui

    2014-01-01

    Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections; however, the mechanism of regulation remains obscure. Here we report that BswR, a xenobiotic response element-type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix motif in the N terminus and two helices (α6 and α7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (α6 and α7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis of BswR homodimer, we proposed a BswR–DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ. PMID:24497189

  4. Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections.

    PubMed

    Baelo, Aida; Levato, Riccardo; Julián, Esther; Crespo, Anna; Astola, José; Gavaldà, Joan; Engel, Elisabeth; Mateos-Timoneda, Miguel Angel; Torrents, Eduard

    2015-07-10

    Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections. PMID:25913364

  5. Psl trails guide exploration and microcolony formation in early P. aeruginosa biofilms

    PubMed Central

    Beckerman, Bernard; Jin, Fan; Gibiansky, Maxsim L.; Harrison, Joe J.; Luijten, Erik; Parsek, Matthew R.; Wong, Gerard C. L.

    2014-01-01

    Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities1-7. Biofilm-forming bacteria such as the opportunistic pathogen7-10 Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors impacting surface adhesion and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as a ‘molecular glue’11-15. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here, we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface via a search-engine based approach16. By combining these techniques with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generate positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Zipf's Law17 indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system18, a ‘rich-get-richer’ mechanism of Psl accumulation that results in a small number of ‘elite’ cells extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl levels determine post-division cell fates and that high local Psl levels ultimately allow ‘elite’ cells to serve as the founding population for initial microcolony development. PMID:23657259

  6. Activation of pulmonary and lymph node dendritic cells during chronic Pseudomonas aeruginosa lung infection in mice.

    PubMed

    Damlund, Dina Silke Malling; Christophersen, Lars; Jensen, Peter Østrup; Alhede, Morten; Høiby, Niels; Moser, Claus

    2016-06-01

    The majority of cystic fibrosis (CF) patients acquire chronic Pseudomonas aeruginosa lung infection, resulting in increased mortality and morbidity. The chronic P. aeruginosa lung infection is characterized by bacteria growing in biofilm surrounded by polymorphonuclear neutrophils (PMNs). However, the infection is not eradicated and the inflammatory response leads to gradual degradation of the lung tissue. In CF patients, a Th2-dominated adaptive immune response with a pronounced antibody response is correlated with poorer outcome. Dendritic cells (DCs) are crucial in bridging the innate immune system with the adaptive immune response. Once activated, the DCs deliver a set of signals to uncommitted T cells that induce development, such as expansion of regulatory T cells and polarization of Th1, Th2 or Th17 subsets. In this study, we characterized DCs in lungs and regional lymph nodes in BALB/c mice infected using intratracheal installation of P. aeruginosa embedded in seaweed alginate in the lungs. A significantly elevated concentration of DCs was detected earlier in the lungs than in the regional lymph nodes. To evaluate whether the chronic P. aeruginosa lung infection leads to activation of DCs, costimulatory molecules CD80 and CD86 were analyzed. During infection, the DCs showed significant elevation of CD80 and CD86 expression in both the lungs and the regional lymph nodes. Interestingly, the percentage of CD86-positive cells was significantly higher than the percentage of CD80-positive cells in the lymph nodes. In addition, cytokine production from Lipopolysaccharides (LPS)-stimulated DCs was analyzed demonstrating elevated production of IL-6, IL-10 and IL-12. However, production of IL-12 was suppressed earlier than IL-6 and IL-10. These results support that DCs are involved in skewing of the Th1/Th2 balance in CF and may be a possible treatment target. PMID:27009697

  7. Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1

    PubMed Central

    Yang, Wen; Li, Kan; Bai, Yuwei; Xu, Yueyang; Jin, Jin; Wang, Yingying; Bartlam, Mark

    2015-01-01

    Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation. PMID:25909591

  8. Synthesis of multiple Pseudomonas aeruginosa biofilm matrix exopolysaccharides is post-transcriptionally regulated

    PubMed Central

    Ma, Luyan; Wang, Juan; Wang, Shiwei; Anderson, Erin M.; Lam, Joseph S.; Parsek, Matthew R.; Wozniak, Daniel J.

    2015-01-01

    Summary Exopolysaccharide is a critical biofilm matrix component, yet little is known about how the synthesis of multiple exopolysaccharides is regulated. Pseudomonas aeruginosa can produce several biofilm matrix exopolysaccharides that include alginate, Psl and Pel. Here we demonstrated that AlgC, a key enzyme that provides sugar precursors for the synthesis of alginate and lipopolysaccharides (LPS) is also required for both Psl and Pel production. We showed that forced-synthesis of Psl in alginate-producing mucoid bacteria reduced alginate production but this was not due to transcription of the alginate biosynthesis-operon. Likewise, when either alginate or Psl were overproduced, levels of B-band LPS decreased. Induction of Pel resulted in a reduction of Psl levels. Because the effects of reduced exopolysaccharide synthesis when another is overproduced didn’t appear to be regulated at the transcriptional level, this suggests that the biosynthesis pathways of Psl, Pel, alginate, and LPS compete for common sugar precursors. As AlgC is the only enzyme that provides precursors for each of these exopolysaccharides, we propose that AlgC is a key checkpoint enzyme that coordinates the total amount of exopolysaccharide biosynthesis by controlling sugar precursor pool. Our data also provide a plausible strategy that P. aeruginosa utilizes to modulate its biofilm matrix exopolysaccharides. PMID:22513190

  9. Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.

    PubMed

    Vidya, P; Smith, L; Beaudoin, T; Yau, Y C; Clark, S; Coburn, B; Guttman, D S; Hwang, D M; Waters, V

    2016-01-01

    Early eradication treatment with inhaled tobramycin is successful in the majority of children with cystic fibrosis (CF) with incident Pseudomonas aeruginosa infection. However, in 10-40 % of cases, eradication fails and the reasons for this are poorly understood. The purpose of this study was to determine whether specific microbial characteristics could explain eradication treatment failure. This was a cross-sectional study of CF patients (aged 0-18 years) with incident P. aeruginosa infection from 2011 to 2014 at the Hospital for Sick Children, Toronto, Canada. Phenotypic assays were done on all incident P. aeruginosa isolates, and eradicated and persistent isolates were compared using the Mann-Whitney test or the two-sided Chi-square test. A total of 46 children with CF had 51 incident P. aeruginosa infections. In 72 % (33/46) of the patients, eradication treatment was successful, while 28 % failed eradication therapy. Persistent isolates were less likely to be motile, with significantly less twitch motility (p=0.001), were more likely to be mucoid (p=0.002), and more likely to have a tobramycin minimum inhibitory concentration (MIC) ≥ 128 μg/mL (p=0.02) compared to eradicated isolates. Although biofilm production was similar, there was a trend towards more persistent isolates with deletions in quorum-sensing genes compared with eradicated isolates (p=0.06). Initial acquisition of P. aeruginosa with characteristics of chronic infection is associated with failure of eradication treatment. PMID:26492874

  10. Mechanics governs single-cell signaling and multi-cell robustness in biofilm infections

    NASA Astrophysics Data System (ADS)

    Gordon, Vernita

    In biofilms, bacteria and other microbes are embedded in extracellular polymers (EPS). Multiple types of EPS can be produced by a single bacterial strain - the reasons for this redundancy are not well-understood. Our work suggests that different polymers may confer distinct mechanical benefits. Our model organism is Pseudomonas aeruginosa, an opportunistic human pathogen that forms chronic biofilm infections associated with increased antibiotic resistance and evasion of the immune defense. Biofilms initiate when bacteria attach to a surface, sense the surface, and change their gene expression. Changes in gene expression are regulated by a chemical signal, cyclic-di-GMP. We find that one EPS material, called ``PEL,'' enhances surface sensing by increasing mechanical coupling of single bacteria to the surface. Measurements of bacterial motility suggest that PEL may increase frictional interactions between the surface and the bacteria. Consistent with this, we show that bacteria increase cyclic-di-GMP signaling in response to mechanical shear stress. Mechanosensing has long been known to be important to the function of cells in higher eukaryotes, but this is one of only a handful of studies showing that bacteria can sense and respond to mechanical forces. For the mature biofilm, the embedding polymer matrix can protect bacteria both chemically and mechanically. P. aeruginosa infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of another EPS material, alginate, is well-known to tend to increase over time in CF infections. Alginate chemically protects biofilms, but also makes them softer and weaker. Recently, it is being increasingly recognized that bacteria in chronic CF infections also evolve to increase PSL production. We use oscillatory bulk rheology to determine the unique contributions of EPS materials to biofilm mechanics. Unlike alginate, increased PSL stiffens biofilms. Increasing both

  11. Egg wax from the cattle tick Rhipicephalus (Boophilus) microplus inhibits Pseudomonas aeruginosa biofilm.

    PubMed

    Zimmer, Karine R; Macedo, Alexandre J; Nicastro, Gianlucca G; Baldini, Regina L; Termignoni, Carlos

    2013-09-01

    Rhipicephalus (Boophilus) microplus is constantly challenged during its life cycle by microorganisms present in their hosts or in the environment. Tick eggs may be especially vulnerable to environmental conditions because they are exposed to a rich and diverse microflora in the soil. Despite being oviposited in such hostile sites, tick eggs remain viable, suggesting that the egg surface has defense mechanisms against opportunistic and/or pathogenic organisms. R. microplus engorged females deposit a superficial wax layer onto their eggs during oviposition. This egg wax is essential for preventing desiccation as well as acting as a barrier against attack by microorganisms. In this study, we report the detection of anti-biofilm activity of R. microplus egg wax against Pseudomonas aeruginosa PA14. Genes involved in the functions of production and maintenance of the biofilm extracellular matrix, pelA and cdrA, respectively, were markedly downregulated by a tick egg-wax extract. Moreover, this extract strongly inhibited fliC gene expression. Instead of a compact extracellular matrix, P. aeruginosa PA14 treated with egg-wax extract produces a fragile one. Also, the colony morphology of cells treated with egg-wax extract appears much paler and brownish, instead of the bright purple characteristic of normal colonies. Swarming motility was also inhibited by treatment with the egg-wax extract. The inhibition of P. aeruginosa biofilm does not seem to depend on inhibition of the quorum sensing system since mRNA levels of the 3 regulators of this system were not inhibited by egg-wax extract. PMID:23583751

  12. Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

    PubMed Central

    Gil, Carmen; Solano, Cristina; Burgui, Saioa; Latasa, Cristina; García, Begoña; Toledo-Arana, Alejandro

    2014-01-01

    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. PMID:24343648

  13. Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm

    PubMed Central

    Baird, Fiona J.; Wadsworth, Marilyn P.; Hill, Jane E.

    2015-01-01

    Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores – ranging in emission from 435 to 665 nm – applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent. PMID:22587931

  14. Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370.

    PubMed

    Zaitseva, Julia; Granik, Vladimir; Belik, Alexandr; Koksharova, Olga; Khmel, Inessa

    2009-06-01

    Antibacterial drugs in the nitrofuran series, such as nitrofurazone, furazidin, nitrofurantoin and nifuroxazide, as well as the nitric oxide generators sodium nitroprusside and isosorbide mononitrate in concentrations that do not suppress bacterial growth, were shown to increase the capacity of pathogenic bacteria Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370 to form biofilms. At 25-100microg/ml, nitrofurans 2-2.5-fold enhanced biofilm formation of P. aeruginosa PAO1, and NO donors 3-6-fold. For B. cenocepacia 370, the enhancement was 2-5-fold (nitrofurans) and 4.5-fold (sodium nitroprusside), respectively. PMID:19460431

  15. Biofilm-based central line-associated bloodstream infections.

    PubMed

    Yousif, Ammar; Jamal, Mohamed A; Raad, Issam

    2015-01-01

    Different types of central venous catheters (CVCs) have been used in clinical practice to improve the quality of life of chronically and critically ill patients. Unfortunately, indwelling devices are usually associated with microbial biofilms and eventually lead to catheter-related bloodstream infections (CLABSIs).An estimated 250,000-400,000 CLABSIs occur every year in the United States, at a rate of 1.5 per 1,000 CVC days and a mortality rate of 12-25 %. The annual cost of caring for patients with CLABSIs ranges from 296 million to 2.3 billion dollars.Biofilm formation occurs on biotic and abiotic surfaces in the clinical setting. Extensive studies have been conducted to understand biofilm formation, including different biofilm developmental stages, biofilm matrix compositions, quorum-sensing regulated biofilm formation, biofilm dispersal (and its clinical implications), and multi-species biofilms that are relevant to polymicrobial infections.When microbes form a matured biofilm within human hosts through medical devices such as CVCs, the infection becomes resistant to antibiotic treatment and can develop into a chronic condition. For that reason, many techniques have been used to prevent the formation of biofilm by targeting different stages of biofilm maturation. Other methods have been used to diagnose and treat established cases of CLABSI.Catheter removal is the conventional management of catheter associated bacteremia; however, the procedure itself carries a relatively high risk of mechanical complications. Salvaging the catheter can help to minimize these complications.In this article, we provide an overview of microbial biofilm formation; describe the involvement of various genetic determinants, adhesion proteins, organelles, mechanism(s) of biofilm formation, polymicrobial infections, and biofilm-associated infections on indwelling intravascular catheters; and describe the diagnosis, management, and prevention of catheter-related bloodstream infections

  16. Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

    PubMed Central

    Schwarzer, Christian; Fu, Zhu; Patanwala, Maria; Hum, Lauren; Lopez-Guzman, Mirielle; Illek, Beate; Kong, Weidong; Lynch, Susan V.; Machen, Terry E.

    2014-01-01

    Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis CF) patients, a process regulated by quorum sensing molecules including N-(3-oxododecanoyl)-L-homoserine lactone, C12. C12 (10–100 μM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CFΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψmito) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca2+ and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 minutes and were complete in 1–2 hrs. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψmito and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψmito and increases in Cacyto like 10–50 μM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed. PMID:22233488

  17. Mannose-centered aromatic galactoclusters inhibit the biofilm formation of Pseudomonas aeruginosa.

    PubMed

    Ligeour, Caroline; Vidal, Olivier; Dupin, Lucie; Casoni, Francesca; Gillon, Emilie; Meyer, Albert; Vidal, Sébastien; Vergoten, Gérard; Lacroix, Jean-Marie; Souteyrand, Eliane; Imberty, Anne; Vasseur, Jean-Jacques; Chevolot, Yann; Morvan, François

    2015-08-21

    Pseudomonas aeruginosa (PA) is a major public health care issue due to its ability to develop antibiotic resistance mainly through adhesion and biofilm formation. Therefore, targeting the bacterial molecular arsenal involved in its adhesion and the formation of its biofilm appears as a promising tool against this pathogen. The galactose-binding LecA (or PA-IL) has been described as one of the PA virulence factors involved in these processes. Herein, the affinity of three tetravalent mannose-centered galactoclusters toward LecA was evaluated with five different bioanalytical methods: HIA, ELLA, SPR, ITC and DNA-based glycoarray. Inhibitory potential towards biofilms was then assessed for the two glycoclusters with highest affinity towards LecA (Kd values of 157 and 194 nM from ITC measurements). An inhibition of biofilm formation of 40% was found for these galactoclusters at 10 μM concentration. Applications of these macromolecules in anti-bacterial therapy are therefore possible through an anti-adhesive strategy. PMID:26090586

  18. Detection of Bacteria Bearing Resistant Biofilm Forms, by Using the Universal and Specific PCR is Still Unhelpful in the Diagnosis of Periprosthetic Joint Infections

    PubMed Central

    Zegaer, Batool H.; Ioannidis, Anastasios; Babis, George C.; Ioannidou, Vassiliki; Kossyvakis, Athanassios; Bersimis, Sotiris; Papaparaskevas, Joseph; Petinaki, Efthimia; Pliatsika, Paraskevi; Chatzipanagiotou, Stylianos

    2014-01-01

    Intraoperative conventional bacteriological cultures were compared with different polymerase chain reaction (PCR) methods in patients with total joint arthroplasties. The isolated bacteria were investigated for biofilm formation, and the biofilm forming strains, in their planktonic and biofilm forms, were further tested for their antimicrobial resistance against several clinically important antimicrobials. Forty four bone and joint samples were included and classified as infected or non-infected according to standard criteria for periprosthetic hip and knee infections. For the bacteriological diagnosis, conventional culture, two types of universal PCR and species specific PCR for three selected pathogens (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) were applied. Biofilm formation determination was performed by the tissue culture plate method. Antimicrobial susceptibility of the planktonic bacteria was performed by the minimal inhibitory concentration determination and, of the biofilm forms, by the minimal inhibitory concentration for bacterial regrowth from the biofilm. Twenty samples were culture positive, with S. epidermidis, S. aureus, or P. aeruginosa. All PCR methods were very ineffective in detecting only one pathogen. All isolates were biofilm positive and their biofilm forms, were highly resistant. In this study, compared to PCR, culture remains the “gold standard.” The biofilm formation by the causative bacteria and the concomitant manifold increased antimicrobial resistance may explain the clinical failure of treatment in some cases and should be considered in the future for therapeutic planning. PMID:25593905

  19. The exopolysaccharide Psl–eDNA interaction enables the formation of a biofilm skeleton in Pseudomonas aeruginosa

    PubMed Central

    Wang, Shiwei; Liu, Xi; Liu, Hongsheng; Zhang, Li; Guo, Yuan; Yu, Shan; Wozniak, Daniel J.; Ma, Luyan Z.

    2015-01-01

    Summary A hallmark of bacterial biofilms is a self-produced extracellular matrix of exopolysaccharide, extracellular DNA (eDNA) and proteins that hold bacterial cells together in the community. However, interactions among matrix components and how the interactions contribute to the formation of matrix remain unclear. Here, we show the physical interaction between exopolysaccharide Psl and eDNA, the two key biofilm matrix components of the opportunistic pathogen Pseudomonas aeruginosa. The interaction allows the two components to combine to form a web of eDNA–Psl fibres, which resembles a biofilm skeleton in the centre of pellicles to give bacteria structural support and capability against agents targeted on one matrix component. The web of eDNA–Psl fibres was also found in flow-cell biofilms at microcolonies initiation stage. The colocalization of eDNA or Psl fibres with bacterial cell membrane stain suggests that fibre-like eDNA is likely derived from the lysis of dead bacteria in biofilms. Psl can interact with DNA from diverse sources, suggesting that P. aeruginosa has the ability to use DNA of other organisms (such as human neutrophils and other bacterial species) to form its own communities, which might increase the survival of P. aeruginosa in multispecies biofilms or within a human host. PMID:25472701

  20. An in situ Raman spectroscopy-based microfluidic "lab-on-a-chip" platform for non-destructive and continuous characterization of Pseudomonas aeruginosa biofilms.

    PubMed

    Feng, Jinsong; de la Fuente-Núñez, César; Trimble, Michael J; Xu, Jie; Hancock, Robert E W; Lu, Xiaonan

    2015-05-28

    Pseudomonas aeruginosa biofilm was cultivated and characterized in a microfluidic "lab-on-a-chip" platform coupled with confocal Raman microscopy in a non-destructive manner. Biofilm formation could be quantified by this label-free platform and correlated well with confocal laser scanning microscopy. This Raman-microfluidic platform could also discriminate biofilms at different developmental stages. PMID:25929246

  1. Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing

    PubMed Central

    Quick, Joshua; Cumley, Nicola; Wearn, Christopher M; Niebel, Marc; Constantinidou, Chrystala; Thomas, Chris M; Pallen, Mark J; Moiemen, Naiem S; Bamford, Amy; Oppenheim, Beryl; Loman, Nicholas J

    2014-01-01

    Objectives Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition. Study design An observational prospective cohort study. Setting Burns care ward and critical care ward in the UK. Participants Patients with >7% total burns by surface area were recruited into the study. Methods All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates. Results WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment. Conclusions This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a

  2. Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume

    PubMed Central

    Pottier, Laurent; Huet, Joelle; Rabemanantsoa, Christian; Kiendrebeogo, Martin; Andriantsimahavandy, Abel; Rasamindrakotroka, Andry; Stévigny, Caroline; Duez, Pierre; El Jaziri, Mondher

    2015-01-01

    Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms. PMID:26186595

  3. Pathogenesis of mucosal biofilm infections: challenges and progress

    PubMed Central

    Dongari-Bagtzoglou, Anna

    2009-01-01

    Living-tissue biofilms remained unrecognized until very recently, mainly as a result of traditional microbial sampling techniques or histologic processing, which disrupt the spatial organization of the tissue microorganisms. Thus, the biofilm nature of certain mucosal infections was frequently unintentionally missed or disregarded. To a large extent, the study of human tissue biofilms is still in its infancy. However, with the advent of newer methodologies, such as fluorescent in situ hybridization and endoscopic confocal laser scanning microscopy, which combine the identification of microbes with in situ, direct visualization of their relationships with each other and with their substratum, mucosal tissue biofilms are becoming easier to study and, thus, their role in human infections is becoming more apparent. This review summarizes the challenges in the study of tissue biofilms, proposes two inflammation-centered – albeit opposite – pathogenetic models of mucosal tissue biofilm infections and suggests directions for future research and novel therapeutic approaches. PMID:18380602

  4. Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14

    PubMed Central

    Strehmel, Janine; Neidig, Anke; Nusser, Michael; Geffers, Robert; Brenner-Weiss, Gerald

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in P. aeruginosa PA14. A PA4398− mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398− mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398− mutant to the wild-type strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold (P < 0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes, we were able to show that not only pvdQ but also pvdR, fptA, pchA, pchD, and pchH are essential for the normal swarming behavior of P. aeruginosa PA14 and may also contribute to the swarming-deficient phenotype of the PA4398− mutant in addition to elevated c-di-GMP levels. PMID:25501476

  5. Sensor kinase PA4398 modulates swarming motility and biofilm formation in Pseudomonas aeruginosa PA14.

    PubMed

    Strehmel, Janine; Neidig, Anke; Nusser, Michael; Geffers, Robert; Brenner-Weiss, Gerald; Overhage, Joerg

    2015-02-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in P. aeruginosa PA14. APA4398 mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398 mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398 mutant to the wildtype strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold (P<0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes,we were able to show that not only pvdQ but also pvdR, fptA, pchA, pchD, and pchH are essential for the normal swarming behavior of P. aeruginosa PA14 and may also contribute to the swarming-deficient phenotype of the PA4398 mutant in addition to elevated c-di-GMP levels. PMID:25501476

  6. Pseudomonas aeruginosa infection mimicking erythema annulare centrifugum.

    PubMed

    Czechowicz, R T; Warren, L J; Moore, L; Saxon, B

    2001-02-01

    A 3-year-old girl receiving chemotherapy for acute lymphocytic leukaemia developed a rapidly expanding red annular plaque on her thigh, initially without signs of systemic toxicity or local pain. Subsequently she developed Pseudomonas aeruginosa sepsis and purpura at the leading edge of the plaque. Skin biopsy showed an extensive necrotizing vasculitis with numerous Gram-negative bacilli in the blood vessel walls. In immunocompromised individuals, skin biopsy and culture of cutaneous lesions for bacteria and fungi should be considered even in the absence of signs of systemic toxicity or multiple lesions. PMID:11233725

  7. Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia

    PubMed Central

    2012-01-01

    Background Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. Results Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. Conclusions The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising “lead compounds” for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease. PMID:22823964

  8. Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels.

    PubMed

    Kim, Han-Shin; Cha, Eunji; Kim, YunHye; Jeon, Young Ho; Olson, Betty H; Byun, Youngjoo; Park, Hee-Deung

    2016-01-01

    Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications. PMID:27141909

  9. Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels

    PubMed Central

    Kim, Han-Shin; Cha, Eunji; Kim, YunHye; Jeon, Young Ho; Olson, Betty H.; Byun, Youngjoo; Park, Hee-Deung

    2016-01-01

    Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications. PMID:27141909

  10. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa.

    PubMed

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  11. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

    PubMed Central

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  12. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms.

    PubMed

    Singh, P K; Schaefer, A L; Parsek, M R; Moninger, T O; Welsh, M J; Greenberg, E P

    2000-10-12

    The bacterium Pseudomonas aeruginosa permanently colonizes cystic fibrosis lungs despite aggressive antibiotic treatment. This suggests that P. aeruginosa might exist as biofilms--structured communities of bacteria encased in a self-produced polymeric matrix--in the cystic fibrosis lung. Consistent with this hypothesis, microscopy of cystic fibrosis sputum shows that P. aeruginosa are in biofilm-like structures. P. aeruginosa uses extracellular quorum-sensing signals (extracellular chemical signals that cue cell-density-dependent gene expression) to coordinate biofilm formation. Here we found that cystic fibrosis sputum produces the two principal P. aeruginosa quorum-sensing signals; however, the relative abundance of these signals was opposite to that of the standard P. aeruginosa strain PAO1 in laboratory broth culture. When P. aeruginosa sputum isolates were grown in broth, some showed quorum-sensing signal ratios like those of the laboratory strain. When we grew these isolates and PAO1 in a laboratory biofilm model, the signal ratios were like those in cystic fibrosis sputum. Our data support the hypothesis that P. aeruginosa are in a biofilm in cystic fibrosis sputum. Moreover, quorum-sensing signal profiling of specific P. aeruginosa strains may serve as a biomarker in screens to identify agents that interfere with biofilm development. PMID:11048725

  13. Autophagy protects C. elegans against necrosis during Pseudomonas aeruginosa infection

    PubMed Central

    Zou, Cheng-Gang; Ma, Yi-Cheng; Dai, Li-Li; Zhang, Ke-Qin

    2014-01-01

    Autophagy, a conserved pathway that delivers intracellular materials into lysosomes for degradation, is involved in development, aging, and a variety of diseases. Accumulating evidence demonstrates that autophagy plays a protective role against infectious diseases by diminishing intracellular pathogens, including bacteria, viruses, and parasites. However, the mechanism by which autophagy regulates innate immunity remains largely unknown. Here, we show that autophagy is involved in host defense against a pathogenic bacterium Pseudomonas aeruginosa in the metazoan Caenorhabditis elegans. P. aeruginosa infection induces autophagy via a conserved extracellular signal-regulated kinase (ERK). Intriguingly, impairment of autophagy does not influence the intestinal accumulation of P. aeruginosa, but instead induces intestinal necrosis. Inhibition of necrosis results in the survival of autophagy-deficient worms after P. aeruginosa infection. These findings reveal a previously unidentified role for autophagy in protection against necrosis triggered by pathogenic bacteria in C. elegans and implicate that such a function of autophagy may be conserved through the inflammatory response in diverse organisms. PMID:25114220

  14. Overcoming barriers in Pseudomonas aeruginosa lung infections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide.

    PubMed

    d'Angelo, Ivana; Casciaro, Bruno; Miro, Agnese; Quaglia, Fabiana; Mangoni, Maria Luisa; Ungaro, Francesca

    2015-11-01

    Cationic antimicrobial peptides (CAMPs) are very promising in the treatment of multi-drug resistant Pseudomonas aeruginosa lung infections experienced by cystic fibrosis (CF) patients. Nevertheless, there is an urgent need of inhalable formulations able to deliver the intact CAMP in conductive airways and to shield its interactions with airway mucus/bacterial biofilm, thus enhancing CAMP/bacteria interactions. Along these lines, the aim of this work was the design and development of nano-embedded microparticles (NEM) for sustained delivery of CAMPs in the lung. To this purpose, nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) containing a model CAMP, colistin (Col), were produced by emulsion/solvent diffusion technique. Engineering NPs with chitosan (CS) and poly(vinyl alcohol) (PVA) allowed to modulate surface properties and, in so doing, to improve NP transport through artificial CF mucus. In order to achieve a long-term stable dosage form useful for NP inhalation, NPs were spray-dried in different carriers (lactose or mannitol), thus producing NEM. The most promising NEM formulations were selected on the basis of bulk and flow properties, distribution of NPs in the carrier and aerosolization performance upon delivery through a breath-actuated dry powder inhaler. Of note, selected Col-loaded NEM were found to kill P. aeruginosa biofilm and to display a prolonged efficacy in biofilm eradication compared to the free Col. This effect was likely ascribable to the ability of NPs to penetrate into bacterial biofilm, as demonstrated by confocal analysis, and to sustain Col release inside it. Taken all together, our results indicate that adequate engineering of PLGA NPs represents an enticing technological approach to harness novel antimicrobials for P. aeruginosa lung infection, such as CAMPs, especially in CF. PMID:26340361

  15. Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models

    PubMed Central

    Madsen, Jonas S.; Lin, Yu-Cheng; Squyres, Georgia R.; Price-Whelan, Alexa; de Santiago Torio, Ana; Song, Angela; Cornell, William C.; Sørensen, Søren J.

    2015-01-01

    As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities. PMID:26431965

  16. Sodium houttuyfonate inhibits biofilm formation and alginate biosynthesis-associated gene expression in a clinical strain of Pseudomonas aeruginosa in vitro

    PubMed Central

    WU, DA-QIANG; CHENG, HUIJUAN; DUAN, QIANGJUN; HUANG, WEIFENG

    2015-01-01

    The increasing multidrug resistance of Pseudomonas aeruginosa has become a serious public-health problem. In the present study, the inhibitory activities of sodium houttuyfonate (SH) against biofilm formation and alginate production in a clinical strain of P. aeruginosa (AH16) were investigated in vitro using crystal violet dying and standard curve methods, respectively. The cellular morphology of P. aeruginosa treated with SH was observed using a scanning electron microscope. Furthermore, reverse transcription-quantitative polymerase chain reaction was used to identify differences in the expression levels of genes associated with alginate biosynthesis as a result of the SH treatment. The results indicated that SH significantly inhibited biofilm formation, and decreased the levels of the primary biofilm constituent, alginate, in P. aeruginosa AH16 at various stages of biofilm development. In addition, scanning electron microscopy observations demonstrated that SH markedly altered the cellular morphology and biofilm structure of P. aeruginosa. Furthermore, the results from the reverse transcription-quantitative polymerase chain reaction analysis indicated that SH inhibited biofilm formation by mitigating the expression of the algD and algR genes, which are associated with alginate biosynthesis. Therefore, the present study has provided novel insights into the potent effects and underlying mechanisms of SH-induced inhibition of biofilm formation in a clinical strain of P. aeruginosa. PMID:26622388

  17. Genotypic analysis of Pseudomonas aeruginosa isolated from ocular infection.

    PubMed

    Yamaguchi, Satoshi; Suzuki, Takashi; Kobayashi, Takeshi; Oka, Naoko; Ishikawa, Eri; Shinomiya, Hiroto; Ohashi, Yuichi

    2014-07-01

    Pseudomonas aeruginosa is the causative pathogen of keratitis, conjunctivitis, and dacryocystitis. However little is known about their clinical epidemiology in Japan. In this study we investigated the genotypic characterization and serotype of P. aeruginosa isolates from ocular infections. Thirty-four clinical P. aeruginosa isolates were characterized according to infection type, the type III secretion system (TTSS), serotype, and multilocus sequence typing (MLST). We divided the isolates into four clinical infection types as follows: Contact lens (CL)-related keratitis (CL-keratitis; 15 isolates), non CL-related keratitis (non CL-keratitis; 8 isolates), conjunctivitis (7 isolates), and dacryocystitis (4 isolates). Regarding the TTSS classification and serotyping classification, no significant differences were found among the infection types. Two clusters (I, II) and three subclusters (A, B, C) were classified according to MLST. CL-keratitis isolates with exoU positivity were clustered in II-B, and conjunctivitis was clustered in cluster I. Some linkage was found between the genetic background and CL-keratitis or conjunctivitis. PMID:24746897

  18. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix

    PubMed Central

    Jennings, Laura K.; Storek, Kelly M.; Ledvina, Hannah E.; Coulon, Charlène; Marmont, Lindsey S.; Sadovskaya, Irina; Secor, Patrick R.; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J.; Howell, P. Lynne; Parsek, Matthew R.

    2015-01-01

    Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components. PMID:26311845

  19. The role of bacterial biofilms in chronic infections.

    PubMed

    Bjarnsholt, Thomas

    2013-05-01

    Acute infections caused by pathogenic bacteria have been studied extensively for well over 100 years. These infections killed millions of people in previous centuries, but they have been combated effectively by the development of modern vaccines, antibiotics and infection control measures. Most research into bacterial pathogenesis has focused on acute infections, but these diseases have now been supplemented by a new category of chronic infections caused by bacteria growing in slime-enclosed aggregates known as biofilms. Biofilm infections, such as pneumonia in cystic fibrosis patients, chronic wounds, chronic otitis media and implant- and catheter-associated infections, affect millions of people in the developed world each year and many deaths occur as a consequence. In general, bacteria have two life forms during growth and proliferation. In one form, the bacteria exist as single, independent cells (planktonic) whereas in the other form, bacteria are organized into sessile aggregates. The latter form is commonly referred to as the biofilm growth phenotype. Acute infections are assumed to involve planktonic bacteria, which are generally treatable with antibiotics, although successful treatment depends on accurate and fast diagnosis. However, in cases where the bacteria succeed in forming a biofilm within the human host, the infection often turns out to be untreatable and will develop into a chronic state. The important hallmarks of chronic biofilm-based infections are extreme resistance to antibiotics and many other conventional antimicrobial agents, and an extreme capacity for evading the host defences. In this thesis, I will assemble the current knowledge on biofilms with an emphasis on chronic infections, guidelines for diagnosis and treatment of these infections, before relating this to my previous research into the area of biofilms. I will present evidence to support a view that the biofilm lifestyle dominates chronic bacterial infections, where bacterial

  20. Biofilm-dependent airway infections: a role for ambroxol?

    PubMed

    Cataldi, M; Sblendorio, V; Leo, A; Piazza, O

    2014-08-01

    Biofilms are a key factor in the development of both acute and chronic airway infections. Their relevance is well established in ventilator associated pneumonia, one of the most severe complications in critically ill patients, and in cystic fibrosis, the most common lethal genetic disease in Caucasians. Accumulating evidence suggests that biofilms could have also a role in chronic obstructive pulmonary disease and their involvement in bronchiectasis has been proposed as well. When they grow in biofilms, microorganisms become multidrug-resistant. Therefore the treatment of biofilm-dependent airway infections is problematic. Indeed, it still largely based on measures aiming to prevent the formation of biofilms or remove them once that they are formed. Here we review recent evidence suggesting that the mucokinetic drug ambroxol has specific anti-biofilm properties. We also discuss how additional pharmacological properties of this drug could be beneficial in biofilm-dependent airway infections. Specifically, we review the evidence showing that: 1-ambroxol exerts anti-inflammatory effects by inhibiting at multiple levels the activity of neutrophils, and 2-it improves mucociliary clearance by interfering with the activity of airway epithelium ion channels and transporters including sodium/bicarbonate and sodium/potassium/chloride cotransporters, cystic fibrosis transmembrane conductance regulator and aquaporins. As a whole, the data that we review here suggest that ambroxol could be helpful in biofilm-dependent airway infections. However, considering the limited clinical evidence available up to date, further clinical studies are required to support the use of ambroxol in these diseases. PMID:24252805

  1. C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth

    PubMed Central

    Chua, Song Lin; Sivakumar, Krishnakumar; Rybtke, Morten; Yuan, Mingjun; Andersen, Jens Bo; Nielsen, Thomas E.; Givskov, Michael; Tolker-Nielsen, Tim; Cao, Bin; Kjelleberg, Staffan; Yang, Liang

    2015-01-01

    Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO32–) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO32– further increased P. aeruginosa biofilm formation and resistance to TeO32–. P. aeruginosa ΔsadCΔsiaD and PAO1/plac-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO32– exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO32–. Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth. PMID:25992876

  2. Novel inhaled combined antibiotic formulations in the treatment of Pseudomonas aeruginosa airways infections in cystic fibrosis.

    PubMed

    Antoniu, Sabina

    2015-07-01

    In cystic fibrosis, chronic airways infection caused by Pseudomonas aeruginosa can be treated with inhaled antibiotics such as inhaled tobramycin, aztreonam or colistin. However, biofilm formation induced by this bacterium can reduce the effectiveness of such therapies and can contribute to antibiotic resistance. Inhaled antibiotic combination might represent an optimal antibiofilm strategy in this setting. This review discusses the rationale for combining the antibiotics as well as some emerging or existing combinations. Most of the combinations except for fosfomycin/tobramycin are at an early stage of development. The latter combination was found to be effective in Phase II clinical studies and is planned to be tested in Phase III trials. The clinical data on long-term efficacy are currently missing, but the existing evidence as well as the unmet therapeutic need can prompt the further evaluation of such compounds. PMID:25921312

  3. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria*

    PubMed Central

    Valentini, Martina

    2016-01-01

    The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission. PMID:27129226

  4. A confocal Raman microscopy study of the distribution of a carotene-containing yeast in a living Pseudomonas aeruginosa biofilm.

    PubMed

    Sandt, Christophe; Smith-Palmer, Truis; Pink, Judith; Pink, David

    2008-09-01

    The distribution of a carotene-containing yeast (CCY) in a biofilm formed by a small colony variant (SCV) of Pseudomonas aeruginosa PA01 was followed by confocal Raman microspectroscopy (CRM). SCV PA01 and CCY cells were distinguished by their spectral signatures, and the distribution of the overall biomass was monitored by the C-H bending or stretching signal. The distributions of total biomass, PA01, and CCY cells were compared at various times and positions within the biofilm. The distribution of the CCY was very heterogeneous. It was found in the water channels as well as in regions within biofilm colonies. Many of the yeast cells observed within the biofilm colonies under conditions of low or stopped flow were removed when medium was flowing, suggesting that the yeast was not held in the matrix as tightly as were the bacteria. PMID:18801236

  5. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria.

    PubMed

    Valentini, Martina; Filloux, Alain

    2016-06-10

    The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission. PMID:27129226

  6. Decreased Pseudomonas aeruginosa biofilm formation on nanomodified endotracheal tubes: a dynamic lung model

    PubMed Central

    Machado, Mary C; Webster, Thomas J

    2016-01-01

    Ventilator-associated pneumonia (VAP) is a serious complication of mechanical ventilation that has been shown to be associated with increased mortality rates and medical costs in the pediatric intensive care unit. Currently, there is no cost-effective solution to the problems posed by VAP. Endotracheal tubes (ETTs) that are resistant to bacterial colonization and that inhibit biofilm formation could provide a novel solution to the problems posed by VAP. The objective of this in vitro study was to evaluate differences in the growth of Pseudomonas aeruginosa on unmodified polyvinyl chloride (PVC) ETTs and on ETTs etched with a fungal lipase, Rhizopus arrhizus, to create nanoscale surface features. These differences were evaluated using an in vitro model of the pediatric airway to simulate a ventilated patient in the pediatric intensive care unit. Each experiment was run for 24 hours and was supported by computational models of the ETT. Dynamic conditions within the ETT had an impact on the location of bacterial growth within the tube. These conditions also quantitatively affected bacterial growth especially within the areas of tube curvature. Most importantly, experiments in the in vitro model revealed a 2.7 log reduction in the number (colony forming units/mL) of P. aeruginosa on the nanoroughened ETTs compared to the untreated PVC ETTs after 24 hours. This reduction in total colony forming units/mL along the x-axis of the tube was similar to previous studies completed for Staphylococcus aureus. Thus, this dynamic study showed that lipase etching can create surface features of nanoscale roughness on PVC ETTs that decrease bacterial attachment of P. aeruginosa without the use of antibiotics and may provide clinicians with an effective and inexpensive tool to combat VAP. PMID:27563242

  7. Decreased Pseudomonas aeruginosa biofilm formation on nanomodified endotracheal tubes: a dynamic lung model.

    PubMed

    Machado, Mary C; Webster, Thomas J

    2016-01-01

    Ventilator-associated pneumonia (VAP) is a serious complication of mechanical ventilation that has been shown to be associated with increased mortality rates and medical costs in the pediatric intensive care unit. Currently, there is no cost-effective solution to the problems posed by VAP. Endotracheal tubes (ETTs) that are resistant to bacterial colonization and that inhibit biofilm formation could provide a novel solution to the problems posed by VAP. The objective of this in vitro study was to evaluate differences in the growth of Pseudomonas aeruginosa on unmodified polyvinyl chloride (PVC) ETTs and on ETTs etched with a fungal lipase, Rhizopus arrhizus, to create nanoscale surface features. These differences were evaluated using an in vitro model of the pediatric airway to simulate a ventilated patient in the pediatric intensive care unit. Each experiment was run for 24 hours and was supported by computational models of the ETT. Dynamic conditions within the ETT had an impact on the location of bacterial growth within the tube. These conditions also quantitatively affected bacterial growth especially within the areas of tube curvature. Most importantly, experiments in the in vitro model revealed a 2.7 log reduction in the number (colony forming units/mL) of P. aeruginosa on the nanoroughened ETTs compared to the untreated PVC ETTs after 24 hours. This reduction in total colony forming units/mL along the x-axis of the tube was similar to previous studies completed for Staphylococcus aureus. Thus, this dynamic study showed that lipase etching can create surface features of nanoscale roughness on PVC ETTs that decrease bacterial attachment of P. aeruginosa without the use of antibiotics and may provide clinicians with an effective and inexpensive tool to combat VAP. PMID:27563242

  8. Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa

    PubMed Central

    Matsuyama, Bruno Y.; Krasteva, Petya V.; Baraquet, Claudine; Harwood, Caroline S.; Sondermann, Holger; Navarro, Marcos V. A. S.

    2016-01-01

    Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ54-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ’s AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP–complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs. PMID:26712005

  9. Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa.

    PubMed

    Matsuyama, Bruno Y; Krasteva, Petya V; Baraquet, Claudine; Harwood, Caroline S; Sondermann, Holger; Navarro, Marcos V A S

    2016-01-12

    Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs. PMID:26712005

  10. Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa.

    PubMed

    Mauline, L; Gressier, M; Roques, C; Hammer, P; Ribeiro, S J L; Caiut, J M A; Menu, M-J

    2013-01-01

    Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium(II) complex. The surface properties of the silica particles were designed by reaction with amino-organosilanes, quaternary ammonium-organosilanes, carboxylate-organosilanes and hexamethyldisilazane. BSNPs were characterized extensively by DRIFT, (13)C and (29)Si solid state NMR, XPS, and photoluminescence. Zeta potential and contact angle measurements exhibited various surface properties (hydrophilic/hydrophobic balance and electric charge) according to the functional groups. Confocal laser scanning microscopy (CLSM) measurements showed that the spatial distribution of these nanoparticles inside a biofilm of Pseudomonas aeruginosa PAO1 depends more on their hydrophilic/hydrophobic characteristics than on their size. CLSM observations using two nanosized particles (25 and 68 nm) suggest that narrow diffusion paths exist through the extracellular polymeric substances matrix. PMID:23805884

  11. An investigation of Pseudomonas aeruginosa biofilm growth on novel nanocellulose fibre dressings.

    PubMed

    Powell, Lydia C; Khan, Saira; Chinga-Carrasco, Gary; Wright, Chris J; Hill, Katja E; Thomas, David W

    2016-02-10

    Nanocellulose from wood is a novel biomaterial, which is highly fibrillated at the nanoscale. This affords the material a number of advantages, including self-assembly, biodegradability and the ability to absorb and retain moisture, which highlights its potential usefulness in clinical wound-dressing applications. In these in vitro studies, the wound pathogen Pseudomonas aeruginosa PAO1 was used to assess the ability of two nanocellulose materials to impair bacterial growth (<48 h). The two nanocelluloses had a relatively small fraction of residual fibres (<4%) and thus a large fraction of nanofibrils (widths <20 nm). Scanning electron microscopy and confocal laser scanning microscopy imaging demonstrated impaired biofilm growth on the nanocellulose films and increased cell death when compared to a commercial control wound dressing, Aquacel(®). Nanocellulose suspensions inhibited bacterial growth, whilst UV-vis spectrophotometry and laser profilometry also revealed the ability of nanocellulose to form smooth, translucent films. Atomic force microscopy studies of the surface properties of nanocellulose demonstrated that PAO1 exhibited markedly contrasting morphology when grown on the nanocellulose film surfaces compared to an Aquacel(®) control dressing (p<0.05). This study highlights the potential utility of these biodegradable materials, from a renewable source, for wound dressing applications in the prevention and treatment of biofilm development. PMID:26686120

  12. Nitric Oxide Signaling in Pseudomonas aeruginosa Biofilms Mediates Phosphodiesterase Activity, Decreased Cyclic Di-GMP Levels, and Enhanced Dispersal▿ †

    PubMed Central

    Barraud, Nicolas; Schleheck, David; Klebensberger, Janosch; Webb, Jeremy S.; Hassett, Daniel J.; Rice, Scott A.; Kjelleberg, Staffan

    2009-01-01

    Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO. PMID:19801410

  13. Application of Dual Inhibition Concept within Looped Autoregulatory Systems toward Antivirulence Agents against Pseudomonas aeruginosa Infections.

    PubMed

    Thomann, Andreas; de Mello Martins, Antonio G G; Brengel, Christian; Empting, Martin; Hartmann, Rolf W

    2016-05-20

    Pseudomonas aeruginosa quorum-sensing (QS) is a sophisticated network of genome-wide regulation triggered in response to population density. A major component is the self-inducing pseudomonas quinolone signal (PQS) QS system that regulates the production of several nonvital virulence- and biofilm-related determinants. Hence, QS circuitry is an attractive target for antivirulence agents with lowered resistance development potential and a good model to study the concept of polypharmacology in autoloop-regulated systems per se. Based on the finding that a combination of PqsR antagonist and PqsD inhibitor synergistically lowers pyocyanin, we have developed a dual-inhibitor compound of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS). In vitro, this compound markedly reduced virulence factor production and biofilm formation accompanied by a diminished content of extracellular DNA (eDNA). Additionally, coadministration with ciprofloxacin increased susceptibility of PA14 to antibiotic treatment under biofilm conditions. Finally, disruption of pathogenicity mechanisms was also assessed in vivo, with significantly increased survival of challenged larvae in a Galleria mellonella infection model. Favorable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, the ability to address two targets of the PQS autoinduction cycle at the same time with a single compound holds great promise in achieving enhanced synergistic cellular effects while potentially lowering rates of resistance development. PMID:26882081

  14. Pseudomonas aeruginosa outcompetes other bacteria in the manifestation and maintenance of a biofilm in polyvinylchloride tubing as used in dental devices.

    PubMed

    Ammann, Christoph Gert; Nagl, Markus; Nogler, Michael; Coraça-Huber, Débora Cristina

    2016-05-01

    In a PVC tube as a model system for dental devices, Pseudomonas aeruginosa outcompetes Staphylococcus aureus and Klebsiella pneumoniae for the biofilm formation. P. aeruginosa has advantage over the other strains due to higher tolerance for low-nutrient situations or direct killing by the production of soluble factors like pyocyanin. PMID:26980595

  15. Synergistic antibiofilm efficacy of various commercial antiseptics, enzymes and EDTA: a study of Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

    PubMed

    Lefebvre, Elodie; Vighetto, Christophe; Di Martino, Patrick; Larreta Garde, Véronique; Seyer, Damien

    2016-08-01

    A multistep strategy was used to generate a combined antibiofilm treatment that could efficiently decrease the biomass of dense biofilms (≥6 × 10(7) CFU/cm(2)). Several compounds that exhibited activity against various targets were tested individually and in combination to search for possible synergistic effects. First, the antibiofilm activity of various commercially available antiseptics was tested on Pseudomonas aeruginosa and Staphylococcus aureus. Second, antiseptics were mixed with ethylene diamine tetra-acetic acid (EDTA), which is an ion chelator that can disturb biofilm organisation, and additive effects on biofilm biomass degradation were found for both strains. Then, enzymes with the ability to destabilise the biofilm matrix by hydrolysing either its proteins or its polysaccharides were used; as expected, they did not decrease bacterial viability but were revealed as efficient biomass reducers. The combination of antiseptics, EDTA and proteases, all at low concentrations, revealed a synergistic effect leading to total eradication of dense biofilms both of P. aeruginosa and S. aureus. PMID:27424598

  16. Current Hypotheses in Cardiac Surgery: Biofilm in Infective Endocarditis.

    PubMed

    Elgharably, Haytham; Hussain, Syed T; Shrestha, Nabin K; Blackstone, Eugene H; Pettersson, Gösta B

    2016-01-01

    Despite recent advances in diagnostics and treatments, infective endocarditis is still associated with substantial morbidity and mortality. Even prolonged courses of broad-spectrum antimicrobials often fail to eradicate the infection, making surgical intervention necessary in many cases. In this review, we present recent advances in molecular microbiology techniques that have uncovered a plausible explanation for this resistance to treatment: the recently discovered social behavior of some microbes, in which colonies form a nearly impenetrable barrier around themselves called a biofilm. These biofilm structures isolate the colony from the body׳s immune response and antimicrobial drugs. We also present current thinking about possible ways biofilms can be destroyed. PMID:27568136

  17. Biofilms

    PubMed Central

    van Hoek, Monique L

    2013-01-01

    Our understanding of the virulence and pathogenesis of Francisella spp. has significantly advanced in recent years, including a new understanding that this organism can form biofilms. What is known so far about Francisella spp. biofilms is summarized here and future research questions are suggested. The molecular basis of biofilm production has begun to be studied, especially the role of extracellular carbohydrates and capsule, quorum sensing and two-component signaling systems. Further work has explored the contribution of amoebae, pili, outer-membrane vesicles, chitinases, and small molecules such as c-di-GMP to Francisella spp. biofilm formation. A role for Francisella spp. biofilm in feeding mosquito larvae has been suggested. As no strong role in virulence has been found yet, Francisella spp. biofilm formation is most likely a key mechanism for environmental survival and persistence. The significance and importance of Francisella spp.’s biofilm phenotype as a critical aspect of its microbial physiology is being developed. Areas for further studies include the potential role of Francisella spp. biofilms in the infection of mammalian hosts and virulence regulation. PMID:24225421

  18. Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFκB signal-transduction pathways

    PubMed Central

    Luo, Jing; Kong, Jin-liang; Dong, Bi-ying; Huang, Hong; Wang, Ke; Wu, Li-hong; Hou, Chang-chun; Liang, Yue; Li, Bing; Chen, Yi-qiang

    2016-01-01

    Burgeoning antibiotic resistance and unfavorable outcomes of inflammatory injury after Pseudomonas aeruginosa infection have necessitated the development of novel agents that not only target quorum sensing (QS) but also combat inflammatory injury with the least risk of resistance. This study aimed to assess the anti-QS and anti-inflammatory activities of baicalein, a traditional herbal medicine that is widely used in the People’s Republic of China, against P. aeruginosa infection. We found that subminimum inhibitory concentrations of baicalein efficiently interfered with the QS-signaling pathway of P. aeruginosa via downregulation of the transcription of QS-regulated genes and the translation of QS-signaling molecules. This interference resulted in the global attenuation of QS-controlled virulence factors, such as motility and biofilm formation, and the secretion into the culture supernatant of extracellular virulence factors, including pyocyanin, LasA protease, LasB elastase, and rhamnolipids. Moreover, we examined the anti-inflammatory activity of baicalein and its mode of action via a P. aeruginosa-infected macrophage model to address its therapeutic effect. Baicalein reduced the P. aeruginosa-induced secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNFα. In addition, baicalein suppressed P. aeruginosa-induced activation of the MAPK and NFκB signal-transduction pathways in cocultured macrophages; this may be the mechanism by which baicalein inhibits the production of proinflammatory cytokines. Therefore, our study demonstrates that baicalein represents a potential treatment for P. aeruginosa infection because it clearly exhibits both antibacterial and anti-inflammatory activities. PMID:26792984

  19. Extensive Reduction of Cell Viability and Enhanced Matrix Production in Pseudomonas aeruginosa PAO1 Flow Biofilms Treated with a d-Amino Acid Mixture

    PubMed Central

    Sanchez, Zoe; Tani, Akio

    2013-01-01

    Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a d-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells. PMID:23220960

  20. Assessment of the working range and effect of sodium dichloroisocyanurate on Pseudomonas aeruginosa biofilms and planktonic cells.

    PubMed

    Morgenthau, Ari; Nicolae, Alexandru M; Laursen, Andrew E; Foucher, Daniel A; Wolfaardt, Gideon M; Hausner, Martina

    2012-01-01

    Sodium dichloroisocyanurate (NaDCC) is a chemical agent that acts against microorganisms in a manner similar to that of sodium hypochlorite by releasing free available chlorine. NaDCC has been approved by the WHO for the emergency treatment of water and by the US EPA for routine treatment of water. Previous studies assessing the effectiveness of NaDCC for the treatment of water implied that NaDCC should have a wide array of disinfecting effects beyond the treatment of planktonic cells in potable water. In this study the biocidal effects of NaDCC against Pseudomonas aeruginosa cells in different growth modes including planktonic cells and biofilms were explored. The data showed that a 60% dilution of the standard NaDCC solution was effective in the treatment of both P. aeruginosa planktonic cells and biofilms. PMID:22263660

  1. The Isolation and the Biofilm Formation of Uropathogens in the Patients with Catheter Associated Urinary Tract Infections (UTIs)

    PubMed Central

    Niveditha, S.; Pramodhini, S.; Umadevi, S.; Kumar, Shailesh; Stephen, Selvaraj

    2012-01-01

    Background Urinary tract infections are the most commonly acquired bacterial infections and they account for an estimated 25-40% of the nosocomial infections. The microbial biofilms pose a public health problem for the persons who require indwelling medical devices, as the microorganisms in the biofilms are difficult to treat with antimicrobial agents. Aims The present study included the isolation and the biofilm formation of the uropathogens in patients with catheter associated urinary tract infections. Methods and Materials This prospective analysis which was carried out over a period of two months, included 50 urine samples from catheterized patients with symptoms of UTI. Following their isolation and identification, all the isolates were subjected to the biofilm detection by the tube adherence method and the Congo Red agar method. Results E.coli was found to be the most frequently isolated uropathogen 35(70%), followed by Klebsiella pneumoniae 8(16%), Pseudomona aeruginosa 2(4%), Acinetobacter spp 1(2%), coagulase negative Staphylococci 3(6%) and Enterococci spp 1(2%). In the current study, 30 (60%) strains were positive in vitro for the biofilm production. Conclusion To conclude, there was significant bacteriuria in all the symptomatic catheterized patients and E.coli was the most frequent isolate. Diabetes (44%) was the most common factor which was associated with the UTIs in the catheterized patients. PMID:23285434

  2. The role of microbial biofilms in prosthetic joint infections.

    PubMed

    Gbejuade, Herbert O; Lovering, Andrew M; Webb, Jason C

    2015-04-01

    Prosthetic joint infection (PJI) still remains a significant problem. In line with the forecasted rise in joint replacement procedures, the number of cases of PJI is also anticipated to rise. The formation of biofilm by causative pathogens is central to the occurrence and the recalcitrance of PJI. The subject of microbial biofilms is receiving increasing attention, probably as a result of the wide acknowledgement of the ubiquity of biofilms in the natural, industrial, and clinical contexts, as well as the notorious difficulty in eradicating them. In this review, we discuss the pertinent issues surrounding PJI and the challenges posed by biofilms regarding diagnosis and treatment. In addition, we discuss novel strategies of prevention and treatment of biofilm-related PJI. PMID:25238433

  3. A Rat Model of Central Venous Catheter to Study Establishment of Long-Term Bacterial Biofilm and Related Acute and Chronic Infections

    PubMed Central

    Chauhan, Ashwini; Lebeaux, David; Decante, Benoit; Kriegel, Irene; Escande, Marie-Christine; Ghigo, Jean-Marc; Beloin, Christophe

    2012-01-01

    Formation of resilient biofilms on medical devices colonized by pathogenic microorganisms is a major cause of health-care associated infection. While in vitro biofilm analyses led to promising anti-biofilm approaches, little is known about their translation to in vivo situations and on host contribution to the in vivo dynamics of infections on medical devices. Here we have developed an in vivo model of long-term bacterial biofilm infections in a pediatric totally implantable venous access port (TIVAP) surgically placed in adult rats. Using non-invasive and quantitative bioluminescence, we studied TIVAP contamination by clinically relevant pathogens, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, and we demonstrated that TIVAP bacterial populations display typical biofilm phenotypes. In our study, we showed that immunocompetent rats were able to control the colonization and clear the bloodstream infection except for up to 30% that suffered systemic infection and death whereas none of the immunosuppressed rats survived the infection. Besides, we mimicked some clinically relevant TIVAP associated complications such as port-pocket infection and hematogenous route of colonization. Finally, by assessing an optimized antibiotic lock therapy, we established that our in vivo model enables to assess innovative therapeutic strategies against bacterial biofilm infections. PMID:22615964

  4. Use of MALDI-TOF mass spectrometry to analyze the molecular profile of Pseudomonas aeruginosa biofilms grown on glass and plastic surfaces.

    PubMed

    Pereira, Flávio D E S; Bonatto, Cínthia C; Lopes, Cláudio A P; Pereira, Alex L; Silva, Luciano P

    2015-09-01

    Biofilms are microbial sessile communities attached to surfaces that are known for causing many medical problems. A bacterial biofilm of clinical relevance is formed by the gram-negative bacteria Pseudomonas aeruginosa. During the formation of a biofilm, the initial adhesion of the cells is of crucial importance, and the characteristics of the contact surface have great influence on this step. In the present study, we aimed to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling as a new methodology to monitor P. aeruginosa biofilm development. Biofilms were grown within polypropylene tubes containing a glass slide, and were harvested after 3, 5, 7, 9, or 12 days of inoculation. Planktonic cells were obtained separately by centrifugation as control. Two independent MALDI-TOF experiments were performed, one by collecting biofilms from both the glass slide and the polypropylene tube internal surface, and the other by acquiring biofilms from these surfaces separately. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to evaluate the morphological progression of the biofilm. The molecular results showed that MALDI profiling is able not only to distinguish between different biofilm stages, but it is also appropriate to indicate when the biofilm cells are released at the dispersion stage, which occurred first on polypropylene surface. Finally, the present study pointed out that MALDI profiling may emerge as a promising tool for the clinical diagnostic and prognostic workup of biofilms formation and control. PMID:26162295

  5. Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections.

    PubMed

    Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W

    2014-01-01

    There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme's electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

  6. General Overview on Nontuberculous Mycobacteria, Biofilms, and Human Infection

    PubMed Central

    Faria, Sonia; Joao, Ines; Jordao, Luisa

    2015-01-01

    Nontuberculous mycobacteria (NTM) are emergent pathogens whose importance in human health has been growing. After being regarded mainly as etiological agents of opportunist infections in HIV patients, they have also been recognized as etiological agents of several infections on immune-competent individuals and healthcare-associated infections. The environmental nature of NTM and their ability to assemble biofilms on different surfaces play a key role in their pathogenesis. Here, we review the clinical manifestations attributed to NTM giving particular importance to the role played by biofilm assembly. PMID:26618006

  7. Sodium Nitrite-Mediated Killing of the Major Cystic Fibrosis Pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under Anaerobic Planktonic and Biofilm Conditions▿

    PubMed Central

    Major, Tiffany A.; Panmanee, Warunya; Mortensen, Joel E.; Gray, Larry D.; Hoglen, Niel; Hassett, Daniel J.

    2010-01-01

    A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO2−) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO2− (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 μg ml−1 NO2−). B. cepacia was particularly sensitive to low concentrations of A-NO2− (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO2− killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of ∼15 mM A-NO2−. Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO2− can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO2− is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO2−, nitric oxide (NO), are dramatically reduced. PMID:20696868

  8. Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa

    SciTech Connect

    Matsuyama, Bruno Y.; Krasteva, Petya V.; Baraquet, Claudine; Harwood, Caroline S.; Sondermann, Holger; Navarro, Marcos V. A. S.

    2015-12-28

    Pseudomonas aeruginosa, an opportunistic pathogen that can cause fatal chronic infections, relies on the intracellular second-messenger c-di-GMP to form robust multicellular biofilms during host tissue colonization. c-di-GMP is sensed directly by the transcription regulator FleQ, which inversely regulates flagellar motility and exopolysaccharide secretion to secure a planktonic to sessile life-form transition. FleQ belongs to the diverse family of AAA+ ATPase enhancer-binding proteins, but how its noncanonical function on transcriptional regulation is controlled by c-di-GMP remains enigmatic. Here, we report structural and functional data that identify an unusual mode of c-di-GMP recognition accompanied by a major quaternary structure reorganization. Our analyses offer a consensus to previous studies and unique insights into the mechanism of action of FleQ and FleQ-like proteins.

  9. Combined treatment of Pseudomonas aeruginosa biofilm with lactoferrin and xylitol inhibits the ability of bacteria to respond to damage resulting from lactoferrin iron chelation.

    PubMed

    Ammons, Mary Cloud B; Ward, Loren S; Dowd, Scot; James, Garth A

    2011-04-01

    With an ageing and ever more obese population, chronic wounds such as diabetic ulcers, pressure ulcers and venous leg ulcers are an increasingly relevant medical concern. Identification of bacterial biofilm contamination as a major contributor to non-healing wounds demands biofilm-targeted strategies to manage chronic wounds. Pseudomonas aeruginosa has been identified as a principal biofilm-forming opportunistic pathogen in chronic wounds. The innate immune molecule lactoferrin and the rare sugar alcohol xylitol have been demonstrated to be co-operatively efficacious against P. aeruginosa biofilms in vitro. Data presented here propose a model for the molecular mechanism behind this co-operative antimicrobial effect. Lactoferrin iron chelation was identified as the primary means by which lactoferrin destabilises the bacterial membrane. By microarray analysis, 183 differentially expressed genes of ≥ 1.5-fold difference were detected. Interestingly, differentially expressed transcripts included the operon encoding components of the pyochelin biosynthesis pathway. Furthermore, siderophore detection verified that xylitol is the component of this novel synergistic treatment that inhibits the ability of the bacteria to produce siderophores under conditions of iron restriction. The findings presented here demonstrate that whilst lactoferrin treatment of P. aeruginosa biofilms results in destabilisation of the bacterial cell membrane though iron chelation, combined treatment with lactoferrin and xylitol inhibits the ability of P. aeruginosa biofilms to respond to environmental iron restriction. PMID:21377840

  10. Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts.

    PubMed

    Lorè, Nicola Ivan; Cigana, Cristina; De Fino, Ida; Riva, Camilla; Juhas, Mario; Schwager, Stephan; Eberl, Leo; Bragonzi, Alessandra

    2012-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host. PMID:22558188

  11. Cystic Fibrosis-Niche Adaptation of Pseudomonas aeruginosa Reduces Virulence in Multiple Infection Hosts

    PubMed Central

    De Fino, Ida; Riva, Camilla; Juhas, Mario; Schwager, Stephan; Eberl, Leo; Bragonzi, Alessandra

    2012-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host. PMID:22558188

  12. Peri-implant infections of oral biofilm etiology.

    PubMed

    Belibasakis, Georgios N; Charalampakis, Georgios; Bostanci, Nagihan; Stadlinger, Bernd

    2015-01-01

    Biofilms are complex microbial communities that grow on various surfaces in nature. The oral micobiota tend to form polymicrobial biofilms, particularly on the hard mineralized surfaces of teeth, which may impact on oral health and disease. They can cause inflammation of the adjacent tooth-supporting (periodontal) tissues, leading to destructive periodontal disease and tooth loss. The emergence of osseointegrated dental implants as a restorative treatment option for replacing missing teeth has also brought along new artificial surfaces within the oral cavity, on which oral bacteria can form biofilms. As in the case of natural teeth, biofilms on implant surfaces may also trigger infection and cause inflammatory destruction of the peri-implant tissue (i.e. peri-implantitis). While there are strong similarities in the composition of the mixed microbial flora between periodontal and peri-implant infections, there are also a few distinctive differences. The immunological events underlying the pathogenesis of peri-implant infections are qualitatively similar, yet more extensive, compared to periodontal infections, resulting in a faster progression of tissue destruction. This chapter summarizes the current knowledge on the microbiology and immunology of peri-implant infections, including findings from the peri-implant crevicular fluid, the inflammatory exudate of the peri-implant tissue. Moreover, it discusses the diagnosis and current approaches for the treatment of oral infections. PMID:25366221

  13. Gene Expression in Pseudomonas aeruginosa: Evidence of Iron Override Effects on Quorum Sensing and Biofilm-Specific Gene Regulation

    PubMed Central

    Bollinger, Nikki; Hassett, Daniel J.; Iglewski, Barbara H.; Costerton, J. William; McDermott, Timothy R.

    2001-01-01

    Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 μM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii

  14. Inoculation density and nutrient level determine the formation of mushroom-shaped structures in Pseudomonas aeruginosa biofilms.

    PubMed

    Ghanbari, Azadeh; Dehghany, Jaber; Schwebs, Timo; Müsken, Mathias; Häussler, Susanne; Meyer-Hermann, Michael

    2016-01-01

    Pseudomonas aeruginosa often colonises immunocompromised patients and the lungs of cystic fibrosis patients. It exhibits resistance to many antibiotics by forming biofilms, which makes it hard to eliminate. P. aeruginosa biofilms form mushroom-shaped structures under certain circumstances. Bacterial motility and the environment affect the eventual mushroom morphology. This study provides an agent-based model for the bacterial dynamics and interactions influencing bacterial biofilm shape. Cell motility in the model relies on recently published experimental data. Our simulations show colony formation by immotile cells. Motile cells escape from a single colony by nutrient chemotaxis and hence no mushroom shape develops. A high number density of non-motile colonies leads to migration of motile cells onto the top of the colonies and formation of mushroom-shaped structures. This model proposes that the formation of mushroom-shaped structures can be predicted by parameters at the time of bacteria inoculation. Depending on nutrient levels and the initial number density of stalks, mushroom-shaped structures only form in a restricted regime. This opens the possibility of early manipulation of spatial pattern formation in bacterial colonies, using environmental factors. PMID:27611778

  15. Inoculation density and nutrient level determine the formation of mushroom-shaped structures in Pseudomonas aeruginosa biofilms

    PubMed Central

    Ghanbari, Azadeh; Dehghany, Jaber; Schwebs, Timo; Müsken, Mathias; Häussler, Susanne; Meyer-Hermann, Michael

    2016-01-01

    Pseudomonas aeruginosa often colonises immunocompromised patients and the lungs of cystic fibrosis patients. It exhibits resistance to many antibiotics by forming biofilms, which makes it hard to eliminate. P. aeruginosa biofilms form mushroom-shaped structures under certain circumstances. Bacterial motility and the environment affect the eventual mushroom morphology. This study provides an agent-based model for the bacterial dynamics and interactions influencing bacterial biofilm shape. Cell motility in the model relies on recently published experimental data. Our simulations show colony formation by immotile cells. Motile cells escape from a single colony by nutrient chemotaxis and hence no mushroom shape develops. A high number density of non-motile colonies leads to migration of motile cells onto the top of the colonies and formation of mushroom-shaped structures. This model proposes that the formation of mushroom-shaped structures can be predicted by parameters at the time of bacteria inoculation. Depending on nutrient levels and the initial number density of stalks, mushroom-shaped structures only form in a restricted regime. This opens the possibility of early manipulation of spatial pattern formation in bacterial colonies, using environmental factors. PMID:27611778

  16. The Pseudomonas aeruginosa CreBC two-component system plays a major role in the response to β-lactams, fitness, biofilm growth, and global regulation.

    PubMed

    Zamorano, Laura; Moyà, Bartolomé; Juan, Carlos; Mulet, Xavier; Blázquez, Jesús; Oliver, Antonio

    2014-09-01

    Pseudomonas aeruginosa is a ubiquitous versatile environmental microorganism with a remarkable ability to grow under diverse environmental conditions. Moreover, P. aeruginosa is responsible for life-threatening infections in immunocompromised and cystic fibrosis patients, as the extraordinary capacity of this pathogen to develop antimicrobial resistance dramatically limits our therapeutic arsenal. Its large genome carries an outstanding number of genes belonging to regulatory systems, including multiple two-component sensor-regulator systems that modulate the response to the different environmental stimuli. Here, we show that one of two systems, designated CreBC (carbon source responsive) and BlrAB (β-lactam resistance), might be of particular relevance. We first identified the stimuli triggering the activation of the CreBC system, which specifically responds to penicillin-binding protein 4 (PBP4) inhibition by certain β-lactam antibiotics. Second, through an analysis of a large comprehensive collection of mutants, we demonstrate an intricate interconnection between the CreBC system, the peptidoglycan recycling pathway, and the expression of the concerning chromosomal β-lactamase AmpC. Third, we show that the CreBC system, and particularly its effector inner membrane protein CreD, plays a major role in bacterial fitness and biofilm development, especially in the presence of subinhibitory concentrations of β-lactams. Finally, global transcriptomics reveals broad regulatory functions of CreBC in basic physiological aspects, particularly anaerobic respiration, in both the presence and absence of antibiotics. Therefore, the CreBC system is envisaged as a potentially interesting target for improving the efficacy of β-lactams against P. aeruginosa infections. PMID:24936599

  17. Characterization of Staphylococcus aureus Biofilm Formation in Urinary Tract Infection

    PubMed Central

    YOUSEFI, Masoud; POURMAND, Mohammad Reza; FALLAH, Fatemeh; HASHEMI, Ali; MASHHADI, Rahil; NAZARI-ALAM, Ali

    2016-01-01

    Background: The aim of this study was to investigate the antibiotic susceptibility pattern as well as the phenotypic and genotypic biofilm formation ability of Staphylococcus aureus isolates from patients with urinary tract infection (UTI). Methods: A total of 39 isolates of S. aureus were collected from patients with UTI. The antibiotic susceptibility patterns of the isolates were determined by the Kirby-Bauer disk-diffusion. We used the Modified Congo red agar (MCRA) and Microtiter plate methods to assess the ability of biofilm formation. All isolates were examined for determination of biofilm related genes, icaA, fnbA, clfA and bap using PCR method. Results: Linezolid, quinupristin/dalfopristin and chloramphenicol were the most effective agents against S. aureus isolates. Overall, 69.2% of S. aureus isolates were biofilm producers. Resistance to four antibiotics such as nitrofurantoin (71.4% vs. 28.6%, P=0.001), tetracycline (57.7% vs. 42.3%, P=0.028), erythromycin and ciprofloxacin (56% vs. 44%, P=0.017) was higher among biofilm producers than non-biofilm producers. The icaA, fnbA and clfA genes were present in all S. aureus isolates. However, bap gene was not detected in any of the isolates. Conclusion: Our findings reinforce the role of biofilm formation in resistance to antimicrobial agents. Trimethoprimsulfamethoxazole and doxycycline may be used as an effective treatment for UTI caused by biofilm producers S. aureus. Our results suggest that biofilm formation is not dependent to just icaA, fnbA, clfA and bap genes harbor in S. aureus strains. PMID:27252918

  18. Pseudomonas aeruginosa Dose-Response and Bathing Water Infection

    EPA Science Inventory

    Pseudomonas aeruginosa is the most commonly identified opportunistic pathogen associated with pool acquired bather disease. To better understand why this microorganism poses this protracted problem we recently appraised P. aeruginosa pool risk management. Much is known about the ...

  19. Pseudomonas aeruginosa Infection in a Group of Captive Humboldt Penguins ( Spheniscus humboldti ).

    PubMed

    Widmer, Dimitri; Ziemssen, Eva; Schade, Benjamin; Kappe, Eva; Schmitt, Ferdinand; Kempf, Hermann; Wibbelt, Gudrun

    2016-06-01

    Nine Humboldt penguins ( Spheniscus humboldti ), between 1 and 1.5 years old and kept at Zoo Dresden, developed local and systemic infections with various opportunistic pathogens within a period of 4 months. Affected birds died peracutely without preceding symptoms or showed various clinical signs, including separation from conspecifics, reduced food intake, lethargy, dyspnea, swelling of the salt glands, and ocular discharge. One bird showed central nervous signs, including seizures. Pathologic examination of deceased birds revealed severe necrotizing inflammation of the mucous membranes and deep structures of the glottis, trachea, nasal sinus, and conchae and granulomatous inflammation of the salt glands. Further findings were airsacculitis, pneumonia, hepatitis, conjunctivitis, and myositis. Pseudomonas aeruginosa was the predominant pathogen in 7 cases. Six penguins died or were euthanatized, whereas 3 penguins that received systemic antibiotic treatment with tobramycin (10 mg/kg IM q24h for 10 days) showed rapid clinical improvement. Insufficient turnover rate of the filtration system, biofilm formation on pipe surfaces, and other factors are assumed to have promoted pathogen buildup in the pool water and subsequent infection. PMID:27315388

  20. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014.

    PubMed

    Høiby, N; Bjarnsholt, T; Moser, C; Bassi, G L; Coenye, T; Donelli, G; Hall-Stoodley, L; Holá, V; Imbert, C; Kirketerp-Møller, K; Lebeaux, D; Oliver, A; Ullmann, A J; Williams, C

    2015-05-01

    Biofilms cause chronic infections in tissues or by developing on the surfaces of medical devices. Biofilm infections persist despite both antibiotic therapy and the innate and adaptive defence mechanisms of the patient. Biofilm infections are characterized by persisting and progressive pathology due primarily to the inflammatory response surrounding the biofilm. For this reason, many biofilm infections may be difficult to diagnose and treat efficiently. It is the purpose of the guideline to bring the current knowledge of biofilm diagnosis and therapy to the attention of clinical microbiologists and infectious disease specialists. Selected hallmark biofilm infections in tissues (e.g. cystic fibrosis with chronic lung infection, patients with chronic wound infections) or associated with devices (e.g. orthopaedic alloplastic devices, endotracheal tubes, intravenous catheters, indwelling urinary catheters, tissue fillers) are the main focus of the guideline, but experience gained from the biofilm infections included in the guideline may inspire similar work in other biofilm infections. The clinical and laboratory parameters for diagnosing biofilm infections are outlined based on the patient's history, signs and symptoms, microscopic findings, culture-based or culture-independent diagnostic techniques and specific immune responses to identify microorganisms known to cause biofilm infections. First, recommendations are given for the collection of appropriate clinical samples, for reliable methods to specifically detect biofilms, for the evaluation of antibody responses to biofilms, for antibiotic susceptibility testing and for improvement of laboratory reports of biofilm findings in the clinical microbiology laboratory. Second, recommendations are given for the prevention and treatment of biofilm infections and for monitoring treatment effectiveness. Finally, suggestions for future research are given to improve diagnosis and treatment of biofilm infections. PMID:25596784

  1. The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development.

    PubMed

    Sakhtah, Hassan; Koyama, Leslie; Zhang, Yihan; Morales, Diana K; Fields, Blanche L; Price-Whelan, Alexa; Hogan, Deborah A; Shepard, Kenneth; Dietrich, Lars E P

    2016-06-21

    Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen. PMID:27274079

  2. Pseudomonas aeruginosa adapts its iron uptake strategies in function of the type of infections.

    PubMed

    Cornelis, Pierre; Dingemans, Jozef

    2013-01-01

    Pseudomonas aeruginosa is a Gram-negative γ-Proteobacterium which is known for its capacity to colonize various niches, including some invertebrate and vertebrate hosts, making it one of the most frequent bacteria causing opportunistic infections. P. aeruginosa is able to cause acute as well as chronic infections and it uses different colonization and virulence factors to do so. Infections range from septicemia, urinary infections, burn wound colonization, and chronic colonization of the lungs of cystic fibrosis patients. Like the vast majority of organisms, P. aeruginosa needs iron to sustain growth. P. aeruginosa utilizes different strategies to take up iron, depending on the type of infection it causes. Two siderophores are produced by this bacterium, pyoverdine and pyochelin, characterized by high and low affinities for iron respectively. P. aeruginosa is also able to utilize different siderophores from other microorganisms (siderophore piracy). It can also take up heme from hemoproteins via two different systems. Under microaerobic or anaerobic conditions, P. aeruginosa is also able to take up ferrous iron via its Feo system using redox-cycling phenazines. Depending on the type of infection, P. aeruginosa can therefore adapt by switching from one iron uptake system to another as we will describe in this short review. PMID:24294593

  3. Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules

    PubMed Central

    Kimyon, Önder; Das, Theerthankar; Ibugo, Amaye I.; Kutty, Samuel K.; Ho, Kitty K.; Tebben, Jan; Kumar, Naresh; Manefield, Mike

    2016-01-01

    Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 μM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 μM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms. PMID:27446013

  4. Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules.

    PubMed

    Kimyon, Önder; Das, Theerthankar; Ibugo, Amaye I; Kutty, Samuel K; Ho, Kitty K; Tebben, Jan; Kumar, Naresh; Manefield, Mike

    2016-01-01

    Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 μM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 μM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms. PMID:27446013

  5. Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches.

    PubMed

    Lund-Palau, Helena; Turnbull, Andrew R; Bush, Andrew; Bardin, Emmanuelle; Cameron, Loren; Soren, Odel; Wierre-Gore, Natasha; Alton, Eric W F W; Bundy, Jacob G; Connett, Gary; Faust, Saul N; Filloux, Alain; Freemont, Paul; Jones, Andy; Khoo, Valerie; Morales, Sandra; Murphy, Ronan; Pabary, Rishi; Simbo, Ameze; Schelenz, Silke; Takats, Zoltan; Webb, Jeremy; Williams, Huw D; Davies, Jane C

    2016-06-01

    Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development. PMID:27175979

  6. Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

    PubMed

    Shakibaie, Mojtaba; Forootanfar, Hamid; Golkari, Yaser; Mohammadi-Khorsand, Tayebe; Shakibaie, Mohammad Reza

    2015-01-01

    The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated. PMID:25175509

  7. Analysis of Bacterial Biofilms on a Cochlear Implant Following Methicillin-Resistant Staphylococcus Aureus Infection

    PubMed Central

    An, Yun Suk; Choi, June; Song, Jae Jun; Chae, Sung Won; Jung, Hak Hyun

    2015-01-01

    To demonstrate biofilm formations on a cochlear implant magnet of a pediatric patient suffering from a methicillin-resistant Staphylococcus aureus (MRSA) infection. The appearance of biofilm colonies was analyzed on different magnet sections. The appearance of MRSA biofilms on the surface of an explanted cochlear implant was analyzed by scanning electron microscopy (SEM), focusing on the pattern of extracellular polymeric substances (EPS) within the biofilms. SEM revealed unique biofilms with a three-dimensional EPS complex and tower-like formations. Biofilm configurations changed from the margin to the center of the magnet. Biofilms were solitary and scattered at the margin; large and plate-like in the center; and stacked in layers, forming towers and water channels, in the middle region. After a MRSA infection, biofilm formations were observed on the surface of a magnet. Bacterial biofilms provide optimal conditions for bacterial growth and antibiotic resistance and can cause intractable infections that lead to device failure. PMID:26771017

  8. The MerR-Like Regulator BrlR Impairs Pseudomonas aeruginosa Biofilm Tolerance to Colistin by Repressing PhoPQ

    PubMed Central

    Chambers, Jacob R.

    2013-01-01

    While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ΔphoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance. PMID:23935054

  9. The urinary antibiotic 5-nitro-8-hydroxyquinoline (Nitroxoline) reduces the formation and induces the dispersal of Pseudomonas aeruginosa biofilms by chelation of iron and zinc.

    PubMed

    Sobke, A; Klinger, M; Hermann, B; Sachse, S; Nietzsche, S; Makarewicz, O; Keller, P M; Pfister, W; Straube, E

    2012-11-01

    Since cations have been reported as essential regulators of biofilm, we investigated the potential of the broad-spectrum antimicrobial and cation-chelator nitroxoline as an antibiofilm agent. Biofilm mass synthesis was reduced by up to 80% at sub-MIC nitroxoline concentrations in Pseudomonas aeruginosa, and structures formed were reticulate rather than compact. In preformed biofilms, viable cell counts were reduced by 4 logs at therapeutic concentrations. Complexation of iron and zinc was demonstrated to underlie nitroxoline's potent antibiofilm activity. PMID:22926564

  10. Biofilm formation by Staphylococcus aureus isolates from skin and soft tissue infections.

    PubMed

    Kwiecinski, Jakub; Kahlmeter, Gunnar; Jin, Tao

    2015-05-01

    Many diseases caused by Staphylococcus aureus are associated with biofilm formation. However, the ability of S. aureus isolates from skin and soft tissue infections to form biofilms has not yet been investigated. We tested 160 isolates from patients with various skin infections for biofilm-forming capacity in different growth media. All the isolates formed biofilms, the extent of which depended on the type of growth medium. The thickest biofilms were formed when both plasma and glucose were present in the broth; in this case, S. aureus incorporated host fibrin into the biofilm's matrix. There were no differences in the biofilm formation between isolates from different types of skin infections, except for a particularly good biofilm formation by isolates from diabetic wounds and a weaker biofilm formation by isolates from impetigo. In conclusion, biofilm formation is a universal behavior of S. aureus isolates from skin infections. In some cases, such as in diabetic wounds, a particularly strong biofilm formation most likely contributes to the chronic and recurrent character of the infection. Additionally, as S. aureus apparently uses host fibrin as part of the biofilm structure, we suggest that plasma should be included more frequently in in vitro biofilm studies. PMID:25586078

  11. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

    PubMed Central

    Irazoqui, Javier E.; Troemel, Emily R.; Feinbaum, Rhonda L.; Luhachack, Lyly G.; Cezairliyan, Brent O.; Ausubel, Frederick M.

    2010-01-01

    The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with

  12. Immobilization of biofilms of Pseudomonas aeruginosa NY3 and their application in the removal of hydrocarbons from highly concentrated oil-containing wastewater on the laboratory scale.

    PubMed

    Nie, Maiqian; Nie, Hongyun; He, Meili; Lin, Yingying; Wang, Lei; Jin, Pengkang; Zhang, SenYuan

    2016-05-15

    To explore the potential of Pseudomonas aeruginosa NY3 for the treatment of highly concentrated crude oil-contaminated water, the immobilization of strain NY3 on the surface of polyurethane foam (PUF), the conditions for using these biofilms and the possibility of recovering the used biofilms were studied. The results demonstrated that the biofilm formation process for strain NY3 was quick and easy. Under optimum conditions, the biomass immobilized on the PUF surface could reach 488.32 mg dry cell/g dry PUF. The results demonstrated that when the degradation time was 12 h, the average oil removal rate in 2 g crude oil/L contaminated water was approximately 90% for 40d. Meanwhile, the biofilms could be recovered for reuse. The recovery ability and the high and steady oil removal rate facilitated the application of the biofilms for the removal of concentrated oil from wastewater. PMID:26963906

  13. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation

    PubMed Central

    Dueholm, Morten S; Søndergaard, Mads T; Nilsson, Martin; Christiansen, Gunna; Stensballe, Allan; Overgaard, Michael T; Givskov, Michael; Tolker-Nielsen, Tim; Otzen, Daniel E; Nielsen, Per H

    2013-01-01

    The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation. PMID:23504942

  14. Pseudomonas aeruginosa uses multiple pathways to acquire iron during chronic infection in cystic fibrosis lungs.

    PubMed

    Konings, Anna F; Martin, Lois W; Sharples, Katrina J; Roddam, Louise F; Latham, Roger; Reid, David W; Lamont, Iain L

    2013-08-01

    Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription-quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF. PMID:23690396

  15. PcrV antibody-antibiotic combination improves survival in Pseudomonas aeruginosa-infected mice.

    PubMed

    Song, Y; Baer, M; Srinivasan, R; Lima, J; Yarranton, G; Bebbington, C; Lynch, S V

    2012-08-01

    The type III secretion system (TTSS) of Pseudomonas aeruginosa, associated with acute infection, facilitates the direct injection of cytotoxins into the host cell cytoplasm. Mab166, a murine monoclonal antibody against PcrV, a protein located at the tip of the injectisome, has demonstrated efficacy against P. aeruginosa infection, resulting in reduced lung injury and increased survival in murine models of infection. We hypothesised that the administration of Mab166 in combination with an antibiotic would further improve the survival of P. aeruginosa-infected mice. A murine model of P. aeruginosa acute infection, three clinically relevant antibiotics (ciprofloxacin, tobramycin and ceftazidime) and the Mab166 antibody were used for this study. Consistently, compared to other treatment groups (antibiotic or antibody administered in isolation), the combination of Mab166 and antibiotic significantly improved the survival of mice infected with three times the lethal dose (LD(90)) of the highly cytotoxic ExoU-secreting strain, PA103. This synergistic effect was primarily due to enhanced bactericidal effect and protection against lung injury, which prevented bacterial dissemination to other organs. Hence, the combination of Mab166 with antibiotic administration provides a new, more effective strategy against P. aeruginosa airway infection, especially when large numbers of highly virulent strains are present. PMID:22187351

  16. IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa.

    PubMed

    Lorè, Nicola Ivan; Cigana, Cristina; Riva, Camilla; De Fino, Ida; Nonis, Alessandro; Spagnuolo, Lorenza; Sipione, Barbara; Cariani, Lisa; Girelli, Daniela; Rossi, Giacomo; Basso, Veronica; Colombo, Carla; Mondino, Anna; Bragonzi, Alessandra

    2016-01-01

    Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a(-/-) or IL-17ra(-/-) mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection. PMID:27189736

  17. IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa

    PubMed Central

    Lorè, Nicola Ivan; Cigana, Cristina; Riva, Camilla; De Fino, Ida; Nonis, Alessandro; Spagnuolo, Lorenza; Sipione, Barbara; Cariani, Lisa; Girelli, Daniela; Rossi, Giacomo; Basso, Veronica; Colombo, Carla; Mondino, Anna; Bragonzi, Alessandra

    2016-01-01

    Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a−/− or IL-17ra−/− mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection. PMID:27189736

  18. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

    PubMed Central

    de la Fuente-Núñez, César; Mansour, Sarah C.; Wang, Zhejun; Jiang, Lucy; Breidenstein, Elena B.M.; Elliott, Melissa; Reffuveille, Fany; Speert, David P.; Reckseidler-Zenteno, Shauna L.; Shen, Ya; Haapasalo, Markus; Hancock, Robert E.W.

    2014-01-01

    Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients. PMID:26221537

  19. Treatment of Pseudomonas aeruginosa-infected orthopedic prostheses with ceftazidime-ciprofloxacin antibiotic combination.

    PubMed

    Brouqui, P; Rousseau, M C; Stein, A; Drancourt, M; Raoult, D

    1995-11-01

    Indwelling device infections are associated with considerable morbidity and extremely high cost. Pseudomonas aeruginosa is the most frequent gram-negative etiologic agent associated with infections of indwelling catheters and foreign body implants. It is generally agreed that eradication of infection in the presence of a foreign body requires removal of the foreign body. Using a combination of ceftazidime and ciprofloxacin, we cured nine of nine patients with P. aeruginosa-infected osteosynthetic material and four of five patients with hip and knee prostheses without removing the foreign material. Follow-up was for a mean of 21 months (range, 6 to 60 months). Some patients experienced minor side effects (arthralgia in one patient and rash in another patient). We conclude that this combination is effective and safe and should be useful in the treatment of P. aeruginosa-infected orthopedic implants. PMID:8585720

  20. Affecting Pseudomonas aeruginosa Phenotypic Plasticity by Quorum Sensing Dysregulation Hampers Pathogenicity in Murine Chronic Lung Infection

    PubMed Central

    Bondí, Roslen; Messina, Marco; De Fino, Ida; Bragonzi, Alessandra; Rampioni, Giordano; Leoni, Livia

    2014-01-01

    In Pseudomonas aeruginosa quorum sensing (QS) activates the production of virulence factors, playing a critical role in pathogenesis. Multiple negative regulators modulate the timing and the extent of the QS response either in the pre-quorum or post-quorum phases of growth. This regulation likely increases P. aeruginosa phenotypic plasticity and population fitness, facilitating colonization of challenging environments such as higher organisms. Accordingly, in addition to the factors required for QS signals synthesis and response, also QS regulators have been proposed as targets for anti-virulence therapies. However, while it is known that P. aeruginosa mutants impaired in QS are attenuated in their pathogenic potential, the effect of mutations causing a dysregulated timing and/or magnitude of the QS response has been poorly investigated so far in animal models of infection. In order to investigate the impact of QS dysregulation on P. aeruginosa pathogenesis in a murine model of lung infection, the QteE and RsaL proteins have been selected as representatives of negative regulators controlling P. aeruginosa QS in the pre- and post-quorum periods, respectively. Results showed that the qteE mutation does not affect P. aeruginosa lethality and ability to establish chronic infection in mice, despite causing a premature QS response and enhanced virulence factors production in test tube cultures compared to the wild type. Conversely, the post-quorum dysregulation caused by the rsaL mutation hampers the establishment of P. aeruginosa chronic lung infection in mice without affecting the mortality rate. On the whole, this study contributes to a better understanding of the impact of QS regulation on P. aeruginosa phenotypic plasticity during the infection process. Possible fallouts of these findings in the anti-virulence therapy field are also discussed. PMID:25420086

  1. Influence of carbapenem resistance on mortality of patients with Pseudomonas aeruginosa infection: a meta-analysis

    PubMed Central

    Liu, Qianqian; Li, Xiaoqing; Li, Wenzhang; Du, Xinmiao; He, Jian-Qing; Tao, Chuanmin; Feng, Yulin

    2015-01-01

    Treatment of infectious diseases caused by the carbapenem-resistant Pseudomonas aeruginosa (CRPA) is becoming more challenging with each passing year. We conducted a meta-analysis to assess the impact of carbapenem resistance on mortality of patients with P. aeruginosa infection. We searched PUBMED, Web of science, EMBASE, Google Scholar and the Cochrane Library up to December 25, 2014, to identify published cohort or case-control studies. 17 studies, including 6660 patients carrying P. aeruginosa, were identified. The pooling analysis indicated that patients infected with CRPA had significantly higher mortality than those infected with carbapenem-susceptible P. aeruginosa (CSPA) (crude OR = 1.64; 95%CI = 1.40, 1.93; adjusted OR = 2.38; 95%CI = 1.53, 3.69). The elevated risk of mortality in patients with CRPA infection was not lessened when stratified by study design, sites of infection, or type of carbapenem, except that the estimate effect vanished in CRPA high-incidence region, South America (crude OR = 1.12; 95%CI = 0.64, 1.99). Begg’s (z = 0.95, p = 0.34) and Egger’s test (t = 1.23, p = 0.24) showed no evidence of publication bias. Our results suggest that carbapenem resistance may increase the mortality of patients with P. aeruginosa infection, whether under univariate or multivariate analysis. PMID:26108476

  2. [The protective activity of 2 normal immunoglobulin preparations for intravenous administration in experimental Pseudomonas aeruginosa infection].

    PubMed

    Vasilev, Ch L; Veleva, K V; Tekelieva, R Kh; Pencheva, P I

    1991-02-01

    The antibody levels in 18 batches of the preparations of human immunoglobulin, Immunovenin and Immunovenin-Intact, for intravenous injection were determined in the enzyme immunoassay with the use of the mixture of P. aeruginosa lipopolysaccharide antigens of seven immunotypes. The average antibody titers in these preparations were identical. The preparations were found to have protective action against P. aeruginosa experimental infection in mice. PMID:1907793

  3. Phage Therapy: a Step Forward in the Treatment of Pseudomonas aeruginosa Infections

    PubMed Central

    Pires, Diana P.; Vilas Boas, Diana; Sillankorva, Sanna

    2015-01-01

    Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and nowadays, a common infection can be fatal. To address this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since Pseudomonas aeruginosa is one of the most common causes of health care-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review further addresses the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy. PMID:25972556

  4. The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection.

    PubMed

    Roberts, Aled E L; Kragh, Kasper N; Bjarnsholt, Thomas; Diggle, Stephen P

    2015-11-20

    We have become increasingly aware that, during infection, pathogenic bacteria often grow in multicellular biofilms that are often highly resistant to antibacterial strategies. In order to understand how biofilms form and contribute to infection, many research groups around the world have heavily used in vitro biofilm systems such as microtitre plate assays and flow cells. Whilst these methods have greatly increased our understanding of the biology of biofilms, it is becoming increasingly apparent that many of our in vitro methods do not accurately represent in vivo conditions. Here we present a systematic review of the most widely used in vitro biofilm systems, and we discuss why they are not always representative of the in vivo biofilms found in chronic infections. We present examples of methods that will help us to bridge the gap between in vitro and in vivo biofilm work so that we can ultimately use our benchside data to improve bedside treatment. PMID:26344834

  5. Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants.

    PubMed

    Phillips, Priscilla L; Yang, Qingping; Davis, Stephen; Sampson, Edith M; Azeke, John I; Hamad, Afifa; Schultz, Gregory S

    2015-08-01

    An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0-3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre-treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time-release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7-log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24-hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release). PMID:24028432

  6. Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection.

    PubMed

    Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra; Visca, Paolo

    2016-08-01

    Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe(3+) uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe(2+) acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe(3+) transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

  7. Nitrosoglutathione generating nitric oxide nanoparticles as an improved strategy for combating Pseudomonas aeruginosa-infected wounds.

    PubMed

    Chouake, Jason; Schairer, David; Kutner, Allison; Sanchez, David A; Makdisi, Joy; Blecher-Paz, Karin; Nacharaju, Parimala; Tuckman-Vernon, Chaim; Gialanella, Phil; Friedman, Joel M; Nosanchuk, Joshua D; Friedman, Adam J

    2012-12-01

    Pseudomonas aeruginosa is a community-acquired, nosocomial pathogen that is an important cause of human morbidity and mortality; it is intrinsically resistant to several antibiotics and is capable of developing resistance to newly developed drugs via a variety of mechanisms. P aeruginosa's ubiquity and multidrug resistance (MDR) warrants the development of innovative methods that overcome its ability to develop resistance. We have previously described a nitric oxide-releasing nanoparticle (NO-np) platform that effectively kills gram-positive and gram-negative organisms in vitro and accelerates clinical recovery in vivo in murine wound and abscess infection models. We have also demonstrated that when glutathione (GSH) is added to NO-np, the nitroso intermediate S-nitrosoglutathione (GSNO) is formed, which has greater activity against P aeruginosa and other gram-negative organisms compared with NO-np alone. In the current study, we evaluate the potential of NO-np to generate GSNO both in vitro and in vivo in a murine excisional wound model infected with an MDR clinical isolate of P aeruginosa. Whereas NO-np alone inhibited P aeruginosa growth in vitro for up to 8 hours, NO-np+GSH completely inhibited P aeruginosa growth for 24 hours. Percent survival in the NO-np+GSH-treated isolates was significantly lower than in the NO-np (36.1% vs 8.3%; P=.004). In addition, NO-np+GSH accelerated wound closure in P aeruginosa-infected wounds, and NO-np+GSH-treated wounds had significantly lower bacterial burden when compared to NO-np-treated wounds (P<.001). We conclude that GSNO is easily generated from our NO-np platform and has the potential to be used as an antimicrobial agent against MDR organisms such as P aeruginosa. PMID:23377518

  8. Efficacy of antiseptics containing povidone-iodine, octenidine dihydrochloride and ethacridine lactate against biofilm formed by Pseudomonas aeruginosa and Staphylococcus aureus measured with the novel biofilm-oriented antiseptics test.

    PubMed

    Junka, Adam; Bartoszewicz, Marzenna; Smutnicka, Danuta; Secewicz, Anna; Szymczyk, Patrycja

    2014-12-01

    Increasing data suggesting that microorganisms in the biofilm form are among the leading agents of persistent infections of chronic wounds require the development of new approaches to treatment. The aim of this article was to compare the efficacy of three commonly used antiseptics using a biofilm-oriented approach. Biofilm-oriented antiseptics test (BOAT), the innovative method, allows to estimate, in a quick and reliable manner, the in vitro activity of working solutions of antiseptics in real contact times against bacteria in the biofilm form and to use the results in the selection of an appropriate antiseptic to treat local infections in the clinical practice. PMID:23445335

  9. Pseudomonas aeruginosa wound infection involves activation of its iron acquisition system in response to fascial contact

    PubMed Central

    Kim, M.; Christley, S.; Khodarev, N. N.; Fleming, I.; Huang, Y.; Chang, E.; Zaborina, O.; Alverdy, J.

    2015-01-01

    Background Wound infections are traditionally thought to occur when microbial burden exceeds the innate clearance capacity of host immune system. Here we introduce the idea that the wound environment itself plays a significant contributory role to wound infection. Methods We developed a clinically relevant murine model of soft tissue infection to explore the role of activation of microbial virulence in response to tissue factors as a mechanism by which pathogenic bacteria cause wound infections. Mice underwent abdominal skin incision and light muscle injury with a crushing forceps versus skin incision alone followed by topical inoculation of P. aeruginosa. Mice were sacrificed on postoperative day 6 and abdominal tissues analyzed for clinical signs of wound infection. To determine if specific wound tissues components induce bacterial virulence, P. aeruginosa was exposed to skin, fascia, and muscle. Results Gross wound infection due to P. aeruginosa was observed to be significantly increased in injured tissues vs non-injured (80% vs 10%) tissues (n=20/group, p<0.0001). Exposure of P. aeruginosa to individual tissue components demonstrated that fascia significantly induced bacterial virulence as judged by the production of pyocyanin, a redox-active phenazine compound known to kill immune cells. Whole genome transcriptional profiling of P. aeruginosa exposed to fascia demonstrated activation of multiple genes responsible for the synthesis of the iron scavenging molecule pyochelin. Conclusion We conclude that wound elements, in particular fascia, may play a significant role in enhancing the virulence of P. aeruginosa and may contribute to the pathogenesis of clinical wound infection. PMID:25807409

  10. Alternative to antibiotics against Pseudomonas aeruginosa: Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.

    PubMed

    Chakotiya, Ankita Singh; Tanwar, Ankit; Narula, Alka; Sharma, Rakesh Kumar

    2016-09-01

    The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 μg ml(-1), respectively. The MBC was found to be 800 and 400 μg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action. PMID:27392698

  11. Impact of new water systems on healthcare-associated colonization or infection with Pseudomonas aeruginosa

    PubMed Central

    Lefebvre, Annick; Quantin, Catherine; Vanhems, Philippe; Lucet, Jean-Christophe; Bertrand, Xavier; Astruc, Karine; Chavanet, Pascal; Aho-Glélé, Ludwig S.

    2016-01-01

    Aim: We aimed to study the impact of new water systems, which were less contaminated with P. aeruginosa, on the incidence of healthcare-associated P. aeruginosa cases (colonizations or infections) in care units that moved to a different building between 2005 and 2014. Methods: Generalized Estimated Equations were used to compare the incidence of P. aeruginosa healthcare-associated cases according to the building. Results: Twenty-nine units moved during the study period and 2,759 cases occurred in these units. No difference was observed when the new building was compared with older buildings overall. Conclusion: Our results did not support our hypothesis of a positive association between water system contamination and the incidence of healthcare-associated P. aeruginosa cases. These results must be confirmed by linking results of water samples and patients’ data. PMID:27274443

  12. (1)H NMR spectroscopy in the diagnosis of Pseudomonas aeruginosa-induced urinary tract infection.

    PubMed

    Gupta, Ashish; Dwivedi, Mayank; Nagana Gowda, G A; Ayyagari, Archana; Mahdi, A A; Bhandari, M; Khetrapal, C L

    2005-08-01

    The utility of (1)H NMR spectroscopy is suggested and demonstrated for the diagnosis of Pseudomonas aeruginosa in urinary tract infection (UTI). The specific property of P. aeruginosa of metabolizing nicotinic acid to 6-hydroxynicotinic acid (6-OHNA) is exploited. The quantity of 6-OHNA produced correlates well with the viable bacterial count. Other common bacteria causing UTI such as Escherichia coli, Klebsiella pneumonia, Enterobacter aerogenes, Acinetobacter baumanii, Proteus mirabilis, Citrobacter frundii, Enterococcus faecalis, Streptococcus gp B and Staphylococcus aureus do not metabolize nicotinic acid under similar conditions. The method provides a single-step documentation of P. aeruginosa qualitatively as well as quantitatively. The NMR method is demonstrated on urine samples from 30 patients with UTI caused by P. aeruginosa. PMID:15759292

  13. Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium

    PubMed Central

    Ferreira, Jose A. G.; Penner, John C.; Moss, Richard B.; Haagensen, Janus A. J.; Clemons, Karl V.; Spormann, Alfred M.; Nazik, Hasan; Cohen, Kevin; Banaei, Niaz; Carolino, Elisabete; Stevens, David A.

    2015-01-01

    Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment. PMID:26252384

  14. Successful treatment of biofilm infections using shock waves combined with antibiotic therapy

    PubMed Central

    Gnanadhas, Divya Prakash; Elango, Monalisha; Janardhanraj, S.; Srinandan, C. S.; Datey, Akshay; Strugnell, Richard A.; Gopalan, Jagadeesh; Chakravortty, Dipshikha

    2015-01-01

    Many bacteria secrete a highly hydrated framework of extracellular polymer matrix on suitable substrates and embed within the matrix to form a biofilm. Bacterial biofilms are observed on many medical devices, endocarditis, periodontitis and lung infections in cystic fibrosis patients. Bacteria in biofilm are protected from antibiotics and >1,000 times of the minimum inhibitory concentration may be required to treat biofilm infections. Here, we demonstrated that shock waves could be used to remove Salmonella, Pseudomonas and Staphylococcus biofilms in urinary catheters. The studies were extended to a Pseudomonas chronic pneumonia lung infection and Staphylococcus skin suture infection model in mice. The biofilm infections in mice, treated with shock waves became susceptible to antibiotics, unlike untreated biofilms. Mice exposed to shock waves responded to ciprofloxacin treatment, while ciprofloxacin alone was ineffective in treating the infection. These results demonstrate for the first time that, shock waves, combined with antibiotic treatment can be used to treat biofilm infection on medical devices as well as in situ infections. PMID:26658706

  15. Protective efficacy of a peptide derived from a potential adhesin of Pseudomonas aeruginosa against corneal infection.

    PubMed

    Yuan, Qing; Wu, Yuting; Wang, Yiqiang; Chen, Lin; Qu, Mingli; Duan, Kangmin; Zhao, Ge

    2016-02-01

    Dissecting the interactions between Pseudomonas aeruginosa and corneal cells is important to identify a novel target for prevention and treatment of Pseudomonas keratitis. The current study began with a peptide identified by phage display, and was to investigate the protective efficacy against P. aeruginosa infection in cornea. The original peptide Pc-E, with high homology to a hypothetical membrane protein (HmpA) in P. aeruginosa, and the derived peptide Pc-EP, with the same sequence as a region in HmpA, were synthesized. Peptide Pc-EP could directly bind to HCEC, stronger than Pc-E, and specifically activate toll-like receptor 5, and thereby significantly induce the production of pro-inflammatory factors, such as IL-1β, IL-6, IFN-γ and IL-17. Moreover, Pc-EP could act as an antagonist to inhibit the adhesion of wild-type P. aeruginosa to HCEC and mouse corneas. No inhibitory effect was observed on the adhesion of the strain loss of HmpA. When compared to the wild-type strain, the adhesion of the hmpA mutant to corneal cells was significantly decreased. Treatment of infected mouse corneas with Pc-EP before infection significantly decreased the bacterial load in the cornea and attenuated the corneal pathology. These results indicate that Pc-EP can be a useful prophylactic agent for P. aeruginosa keratitis. PMID:26500187

  16. Virulence Gene Profiles of Multidrug-Resistant Pseudomonas aeruginosa Isolated From Iranian Hospital Infections

    PubMed Central

    Fazeli, Nastaran; Momtaz, Hassan

    2014-01-01

    Background: The most common hospital-acquired pathogen is Pseudomonas aeruginosa. It is a multidrug resistant bacterium causing systemic infections. Objectives: The present study was carried out in order to investigate the distribution of virulence factors and antibiotic resistance properties of Pseudomonas aeruginosa isolated from various types of hospital infections in Iran. Patients and Methods: Two-hundred and seventeen human infection specimens were collected from Baqiyatallah and Payambaran hospitals in Tehran, Iran. The clinical samples were cultured immediately and samples positive for P. aeruginosa were analyzed for the presence of antibiotic resistance and bacterial virulence genes using PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed using disk diffusion methodology with Müeller–Hinton agar. Results: Fifty-eight out of 127 (45.66%) male infection specimens and 44 out of 90 (48.88%) female infection specimens harbored P. aeruginosa. Also, 65% (in male specimens) and 21% (in female specimens) of respiratory system infections were positive for P. aeruginosa, which was a high rate. The genes encoding exoenzyme S (67.64%) and phospholipases C (45.09%) were the most common virulence genes found among the strains. The incidences of various β-lactams encoding genes, including blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, and blaVEB were 94.11%, 16.66%, 15.68%, 18.62%, 21.56%, and 17.64%, respectively. The most commonly detected fluoroquinolones encoding gene was gyrA (15. 68%). High resistance levels to penicillin (100%), tetracycline (90.19%), streptomycin (64.70%), and erythromycin (43.13%) were observed too. Conclusions: Our findings should raise awareness about antibiotic resistance in hospitalized patients in Iran. Clinicians should exercise caution in prescribing antibiotics, especially in cases of human infections. PMID:25763199

  17. Biofilm initiation and growth of Pseudomonas aeruginosa on 316L stainless steel in low gravity in orbital space flight

    NASA Astrophysics Data System (ADS)

    Todd, Paul; Pierson, Duane L.; Allen, Britt; Silverstein, JoAnn

    The formation of biofilms by water microorganisms such as Pseudomonas aeruginosa in spacecraft water systems has been a matter of concern for long-duration space flight. Crewed spacecraft plumbing includes internal surfaces made of 316L stainless steel. Experiments were therefore undertaken to compare the ability of P. aeruginosa to grow in suspension, attach to stainless steel and to grow on stainless steel in low gravity on the space shuttle. Four categories of cultures were studied during two space shuttle flights (STS-69 and STS-77). Cultures on the ground were held in static horizontal or vertical cylindrical containers or were tumbled on a clinostat and activated under conditions identical to those for the flown cultures. The containers used on the ground and in flight were BioServe Space Technologies’ Fluid Processing Apparatus (FPA), an open-ended test tube with rubber septa that allows robotic addition of bacteria to culture media to initiate experiments and the addition of fixative to conclude experiments. Planktonic growth was monitored by spectrophotometry, and biofilms were characterized quantitatively by epifluorescence and scanning electron microscopy. In these experiments it was found that: (1) Planktonic growth in flown cultures was more extensive than in static cultures, as seen repeatedly in the history of space microbiology, and closely resembled the growth of tumbled cultures. (2) Conversely, the attachment of cells in flown cultures was as much as 8 times that in tumbled cultures but not significantly different from that in static horizontal and vertical cultures, consistent with the notion that flowing fluid reduces microbial attachment. (3) The final surface coverage in 8 days was the same for flown and static cultures but less by a factor of 15 in tumbled cultures, where coverage declined during the preceding 4 days. It is concluded that cell attachment to 316L stainless steel in the low gravity of orbital space flight is similar to that

  18. Shift in Ribonucleotide Reductase Gene Expression in Pseudomonas aeruginosa during Infection ▿ †

    PubMed Central

    Sjöberg, Britt-Marie; Torrents, Eduard

    2011-01-01

    The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients. PMID:21502590

  19. Genomic Variation among Contemporary Pseudomonas aeruginosa Isolates from Chronically Infected Cystic Fibrosis Patients

    PubMed Central

    Chung, Jade C. S.; Becq, Jennifer; Fraser, Louise; Schulz-Trieglaff, Ole; Bond, Nicholas J.; Foweraker, Juliet; Bruce, Kenneth D.; Smith, Geoffrey P.

    2012-01-01

    The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates. PMID:22753054

  20. Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients.

    PubMed

    Jeukens, Julie; Boyle, Brian; Kukavica-Ibrulj, Irena; Ouellet, Myriam M; Aaron, Shawn D; Charette, Steve J; Fothergill, Joanne L; Tucker, Nicholas P; Winstanley, Craig; Levesque, Roger C

    2014-01-01

    Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung. PMID:24505294

  1. Spanish consensus on the prevention and treatment of Pseudomonas aeruginosa bronchial infections in cystic fibrosis patients.

    PubMed

    Cantón, Rafael; Máiz, Luis; Escribano, Amparo; Olveira, Casilda; Oliver, Antonio; Asensio, Oscar; Gartner, Silvia; Roma, Eva; Quintana-Gallego, Esther; Salcedo, Antonio; Girón, Rosa; Barrio, María Isabel; Pastor, María Dolores; Prados, Concepción; Martínez-Martínez, María Teresa; Barberán, José; Castón, Juan José; Martínez-Martínez, Luis; Poveda, José Luis; Vázquez, Carlos; de Gracia, Javier; Solé, Amparo

    2015-03-01

    Pseudomonas aeruginosa is the main pathogen in bronchopulmonary infections in cystic fibrosis (CF) patients. It can only be eradicated at early infection stages while reduction of its bacterial load is the therapeutic goal during chronic infection or exacerbations. Neonatal screening and pharmacokinetic/pharmacodynamic knowledge has modified the management of CF-patients. A culture based microbiological follow-up should be performed in patients with no infection with P.aeruginosa. At initial infection, inhaled colistin (0,5-2MU/tid), tobramycin (300mg/bid) or aztreonam (75mg/tid) with or without oral ciprofloxacin (15-20mg/kg/bid, 2-3weeks) are recommended. In chronic infections, treatment is based on continuous administration of colistin or with a 28-day on-off regimen with tobramycin or aztreonam. During mild-moderate exacerbations oral ciprofloxacin (2-3weeks) can be administered while serious exacerbations must be treated with intravenous combination therapy (beta-lactam with an aminoglycoside or a fluoroquinolone). Future studies will support antibiotic rotation and/or new combination therapies. Epidemiological measures are also recommended to avoid new P.aeruginosa infections and "patient-to-patient transmission" of this pathogen. PMID:25614377

  2. On-Demand Removal of Bacterial Biofilms via Shape Memory Activation.

    PubMed

    Gu, Huan; Lee, Sang Won; Buffington, Shelby Lois; Henderson, James H; Ren, Dacheng

    2016-08-24

    Bacterial biofilms are a major cause of chronic infections and biofouling; however, effective removal of established biofilms remains challenging. Here we report a new strategy for biofilm control using biocompatible shape memory polymers with defined surface topography. These surfaces can both prevent bacterial adhesion and remove established biofilms upon rapid shape change with moderate increase of temperature, thereby offering more prolonged antifouling properties. We demonstrate that this strategy can achieve a total reduction of Pseudomonas aeruginosa biofilms by 99.9% compared to the static flat control. It was also found effective against biofilms of Staphylococcus aureus and an uropathogenic strain of Escherichia coli. PMID:27517738

  3. Microbial diversity in biofilm infections of the urinary tract with the use of sonication techniques.

    PubMed

    Holá, Veronika; Ruzicka, Filip; Horka, Marie

    2010-08-01

    Infections of the urinary tract account for >40% of nosocomial infections; most of these are infections in catheterized patients. Bacterial colonization of the urinary tract and catheters causes not only the particular infection but also a number of complications, for example blockage of catheters with crystallic deposits of bacterial origin, generation of gravels and pyelonephritis. Infections of urinary catheters are only rarely single-species infections. The longer a patient is catheterized, the higher the diversity of biofilm microbial communities. The aims of this study were to investigate the microbial diversity on the catheters and to compare the ability to form biofilm among isolated microbial species. The next aim was to discriminate particular causative agents of infections of the urinary tract and their importance as biofilm formers in the microbial community on the urinary catheter. We examined catheters from 535 patients and isolated 1555 strains of microorganisms. Most of the catheters were infected by three or more microorganisms; only 12.5% showed monomicrobial infection. Among the microorganisms isolated from the urinary catheters, there were significant differences in biofilm-forming ability, and we therefore conclude that some microbial species have greater potential to cause a biofilm-based infection, whereas others can be only passive members of the biofilm community. PMID:20602639

  4. Prosthesis Infections after Orthopedic Joint Replacement: The Possible Role of Bacterial Biofilms

    PubMed Central

    Song, Zhijun; Borgwardt, Lotte; Høiby, Niels; Wu, Hong; Sørensen, Torben Sandberg; Borgwardt, Arne

    2013-01-01

    Prosthesis-related infection is a serious complication for patients after orthopedic joint replacement, which is currently difficult to treat with antibiotic therapy. Consequently, in most cases, removal of the infected prosthesis is the only solution to cure the infection. It is, therefore, important to understand the comprehensive interaction between the microbiological situation and the host immune responses that lead to prosthesis infections. Evidence indicates that prosthesis infections are actually biofilm-correlated infections that are highly resistant to antibiotic treatment and the host immune responses. The authors reviewed the related literature in the context of their clinical experience, and discussed the possible etiology and mechanism leading to the infections, especially problems related to bacterial biofilm, and prophylaxis and treatment of infection, including both microbiological and surgical measures. Recent progress in research into bacterial biofilm and possible future treatment options of prosthesis-related infections are discussed. PMID:23888204

  5. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    PubMed

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  6. The extra-cytoplasmic function sigma factor sigX modulates biofilm and virulence-related properties in Pseudomonas aeruginosa.

    PubMed

    Gicquel, Gwendoline; Bouffartigues, Emeline; Bains, Manjeet; Oxaran, Virginie; Rosay, Thibaut; Lesouhaitier, Olivier; Connil, Nathalie; Bazire, Alexis; Maillot, Olivier; Bénard, Magalie; Cornelis, Pierre; Hancock, Robert E W; Dufour, Alain; Feuilloley, Marc G J; Orange, Nicole; Déziel, Eric; Chevalier, Sylvie

    2013-01-01

    SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the "chaperones and heat shock proteins", "antibiotic resistance and susceptibility", "energy metabolism", "protein secretion/export apparatus", and "secreted factors", and "motility and attachment" classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes. PMID:24260387

  7. The Extra-Cytoplasmic Function Sigma Factor SigX Modulates Biofilm and Virulence-Related Properties in Pseudomonas aeruginosa

    PubMed Central

    Gicquel, Gwendoline; Bouffartigues, Emeline; Bains, Manjeet; Oxaran, Virginie; Rosay, Thibaut; Lesouhaitier, Olivier; Connil, Nathalie; Bazire, Alexis; Maillot, Olivier; Bénard, Magalie; Cornelis, Pierre; Hancock, Robert E. W.; Dufour, Alain; Feuilloley, Marc G. J.; Orange, Nicole; Déziel, Eric; Chevalier, Sylvie

    2013-01-01

    SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the “chaperones and heat shock proteins”, “antibiotic resistance and susceptibility”, “energy metabolism”, “protein secretion/export apparatus”, and “secreted factors”, and “motility and attachment” classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes. PMID:24260387

  8. Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis.

    PubMed

    Wright, Elli A; Di Lorenzo, Valeria; Trappetti, Claudia; Liciardi, Manuele; Orru, Germano; Viti, Carlo; Bronowski, Christina; Hall, Amanda J; Darby, Alistair C; Oggioni, Marco R; Winstanley, Craig

    2015-01-30

    Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections. PMID:25475851

  9. Mimicking the host and its microenvironment in vitro for studying mucosal infections by Pseudomonas aeruginosa

    PubMed Central

    Crabbé, Aurélie; Ledesma, Maria A.; Nickerson, Cheryl A.

    2014-01-01

    Why is a healthy person protected from Pseudomonas aeruginosa infections, while individuals with cystic fibrosis or damaged epithelium are particularly susceptible to this opportunistic pathogen? In order to address this question, it is essential to thoroughly understand the dynamic interplay between the host microenvironment and P. aeruginosa. Therefore, using modeI systems that represent key aspects of human mucosal tissues in health and disease allows recreating in vivo host-pathogen interactions in a physiologically relevant manner. In this review, we discuss how factors of mucosal tissues, such as apical-basolateral polarity, junctional complexes, extracellular matrix proteins, mucus, multicellular complexity (including indigenous microbiota), and other physicochemical factors affect P. aeruginosa pathogenesis and are thus important to mimic in vitro. We highlight in vitro cell and tissue culture model systems of increasing complexity that have been used over the past 35 years to study the infectious disease process of P. aeruginosa, mainly focusing on lung models, and their respective advantages and limitations. Continued improvements of in vitro models based on our expanding knowledge of host microenvironmental factors that participate in P. aeruginosa pathogenesis will help advance fundamental understanding of pathogenic mechanisms and increase the translational potential of research findings from bench to the patient’s bedside. PMID:24737619

  10. Phase II studies of nebulised Arikace in CF patients with Pseudomonas aeruginosa infection

    PubMed Central

    Clancy, J P; Dupont, L; Konstan, M W; Billings, J; Fustik, S; Goss, C H; Lymp, J; Minic, P; Quittner, A L; Rubenstein, R C; Young, K R; Saiman, L; Burns, J L; Govan, J R W; Ramsey, B; Gupta, R

    2013-01-01

    Rationale Arikace is a liposomal amikacin preparation for aerosol delivery with potent Pseudomonas aeruginosa killing and prolonged lung deposition. Objectives To examine the safety and efficacy of 28 days of once-daily Arikace in cystic fibrosis (CF) patients chronically infected with P aeruginosa. Methods 105 subjects were evaluated in double-blind, placebo-controlled studies. Subjects were randomised to once-daily Arikace (70, 140, 280 and 560 mg; n=7, 5, 21 and 36 subjects) or placebo (n=36) for 28 days. Primary outcomes included safety and tolerability. Secondary outcomes included lung function (forced expiratory volume at one second (FEV1)), P aeruginosa density in sputum, and the Cystic Fibrosis Quality of Life Questionnaire—Revised (CFQ-R). Results The adverse event profile was similar among Arikace and placebo subjects. The relative change in FEV1 was higher in the 560 mg dose group at day 28 (p=0.033) and at day 56 (28 days post-treatment, 0.093L±0.203 vs −0.032L±0.119; p=0.003) versus placebo. Sputum P aeruginosa density decreased >1 log in the 560 mg group versus placebo (days 14, 28 and 35; p=0.021). The Respiratory Domain of the CFQ-R increased by the Minimal Clinically Important Difference (MCID) in 67% of Arikace subjects (560 mg) versus 36% of placebo (p=0.006), and correlated with FEV1 improvements at days 14, 28 and 42 (p<0.05). An open-label extension (560 mg Arikace) for 28 days followed by 56 days off over six cycles confirmed durable improvements in lung function and sputum P aeruginosa density (n=49). Conclusions Once-daily Arikace demonstrated acute tolerability, safety, biologic activity and efficacy in patients with CF with P aeruginosa infection. PMID:23749840

  11. In vitro activity of dalbavancin against biofilms of staphylococci isolated from prosthetic joint infections.

    PubMed

    Fernández, Javier; Greenwood-Quaintance, Kerryl E; Patel, Robin

    2016-08-01

    The in vitro activity of dalbavancin was tested against biofilms of 171 staphylococci associated with prosthetic joint infection. Dalbavancin minimum biofilm bactericidal concentration (MBBC) values were: MBBC50 for Staphylococcus aureus and Staphylococcus epidermidis, 1μg/mL; MBBC90 for S. aureus, 2μg/mL; MBBC90 for S. epidermidis, 4μg/mL. PMID:27241369

  12. Effects of Green Tea Compound Epigallocatechin-3-Gallate against Stenotrophomonas maltophilia Infection and Biofilm

    PubMed Central

    Vidigal, Pedrina G.; Müsken, Mathias; Becker, Katrin A.; Häussler, Susanne; Wingender, Jost; Steinmann, Eike; Kehrmann, Jan; Gulbins, Erich; Buer, Jan; Rath, Peter Michael; Steinmann, Jörg

    2014-01-01

    We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF. PMID:24690894

  13. Effects of green tea compound epigallocatechin-3-gallate against Stenotrophomonas maltophilia infection and biofilm.

    PubMed

    Vidigal, Pedrina G; Müsken, Mathias; Becker, Katrin A; Häussler, Susanne; Wingender, Jost; Steinmann, Eike; Kehrmann, Jan; Gulbins, Erich; Buer, Jan; Rath, Peter Michael; Steinmann, Jörg

    2014-01-01

    We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF. PMID:24690894

  14. Production of Quorum Sensing Inhibitors in Growing Onion Bulbs Infected with Pseudomonas aeruginosa E (HQ324110)

    PubMed Central

    Abd-Alla, Mohamed H.; Bashandy, Shymaa R.

    2012-01-01

    Eighteen organic compounds were present in growing onion bulbs cultivar Giza 6 infected with P. aeruginosa, but only fourteen of them are present in dry infected onion bulbs; however, four compounds were missing in dry onion. The missing compounds in dry infected onion bulbs are pantolactone, 4,5-dihydro-4,5-dimethylfuran-2(3H)-one, myristic acid, and linoleic acid. All of them were detected in growing onion (living cell) during Pseudomonas aeruginosa infection, and it is hypothesized that it may be produced by plants and act as defence system. Pantolactone and myristic acid were selected to explore their effects on growth and virulence factors of Pseudomonas aeruginosa. Exogenous application of pantolactone and myristic acid significantly inhibited pyocyanin production, protease, and lipase and polygalacturonase activity but did not have any significant effects on bacterial growth. The inhibition of virulence factors without reduction in bacterial growth may be providing strong support that these chemical molecules are general quorum sensing inhibitors than an antibacterial effect. Disruption of quorum sensing of pathogen indicates that this new approach has potential in fighting bacterial infections in human and plants. PMID:23724316

  15. Increased susceptibility to Pseudomonas aeruginosa infection under hindlimb-unloading conditions

    NASA Technical Reports Server (NTRS)

    Aviles, Hernan; Belay, Tesfaye; Fountain, Kimberly; Vance, Monique; Sonnenfeld, Gerald

    2003-01-01

    It has been reported that spaceflight conditions alter the immune system and resistance to infection [Belay T, Aviles H, Vance M, Fountain K, and Sonnenfeld G. J Allergy Clin Immunol 170: 262-268, 2002; Hankins WR and Ziegelschmid JF. In: Biomedical Results of Apollo. Washington, DC: NASA, 1975, p. 43-81. (NASA Spec. Rep. SP-368)]. Ground-based models, including the hindlimb-unloading model, have become important tools for increasing understanding of how spaceflight conditions can influence physiology. The objective of the present study was to determine the effect of hindlimb unloading on the susceptibility of mice to Pseudomonas aeruginosa infection. Hindlimb-unloaded and control mice were subcutaneously infected with 1 LD50 of P. aeruginosa. Survival, bacterial organ load, and antibody and corticosterone levels were compared among the groups. Hindlimb unloading had detrimental effects for infected mice. Animals in the hindlimb-unloaded group, compared with controls, 1). showed significantly increased mortality and reduced time to death, 2). had increased levels of corticosterone, and 3). were much less able to clear bacteria from the organs. These results suggest that hindlimb unloading may induce the production of corticosterone, which may play a critical role in the modulation of the immune system leading to increased susceptibility to P. aeruginosa infection.

  16. Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

    PubMed

    Beeton, M L; Alves, D R; Enright, M C; Jenkins, A T A

    2015-08-01

    The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses. PMID:26100212

  17. Effects of Chinese medicinal herbs on a rat model of chronic Pseudomonas aeruginosa lung infection.

    PubMed

    Song, Z; Johansen, H K; Moser, C; Høiby, N

    1996-05-01

    The aim of the study was to evaluate the effects of two kinds of Chinese medicinal herbs, Isatis tinctoria L (ITL) and Daphne giraldii Nitsche (DGN), on a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF). Compared to the control group, both drugs were able to reduce the incidence of lung abscess (p < 0.05) and to decrease the severity of the macroscopic pathology in lungs (p < 0.05). In the great majority of the rats, the herbs altered the inflammatory response in the lungs from an acute type inflammation, dominated by polymorphonuclear leukocytes (PMN), to a chronic type inflammation, dominated by mononuclear leukocytes (MN). DGN also improved the clearance of P. aeruginosa from the lungs (p < 0.03) compared with the control group. There were no significant differences between the control group and the two herbal groups with regard to serum IgG and IgA anti-P. aeruginosa sonicate antibodies. However, the IgM concentration in the ITL group was significantly lower than in the control group (p < 0.03). These results suggest that the two medicinal herbs might be helpful to CF patients with chronic P. aeruginosa lung infection, DGN being the most favorable. PMID:8703440

  18. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis.

    PubMed

    Clark, Shawn T; Diaz Caballero, Julio; Cheang, Mary; Coburn, Bryan; Wang, Pauline W; Donaldson, Sylva L; Zhang, Yu; Liu, Mingyao; Keshavjee, Shaf; Yau, Yvonne C W; Waters, Valerie J; Elizabeth Tullis, D; Guttman, David S; Hwang, David M

    2015-01-01

    Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to β-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample. PMID:26047320

  19. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis

    PubMed Central

    Clark, Shawn T.; Diaz Caballero, Julio; Cheang, Mary; Coburn, Bryan; Wang, Pauline W.; Donaldson, Sylva L.; Zhang, Yu; Liu, Mingyao; Keshavjee, Shaf; Yau, Yvonne C.W.; Waters, Valerie J.; Elizabeth Tullis, D.; Guttman, David S.; Hwang, David M.

    2015-01-01

    Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to β-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample. PMID:26047320

  20. From in vitro to in vivo Models of Bacterial Biofilm-Related Infections

    PubMed Central

    Lebeaux, David; Chauhan, Ashwini; Rendueles, Olaya; Beloin, Christophe

    2013-01-01

    The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30–40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These different models have progressively contributed to the current knowledge of biofilm physiology within the host context. While far from a complete understanding of the multiple elements controlling the dynamic interactions between the host and biofilms, we are nowadays witnessing the emergence of promising preventive or curative strategies to fight biofilm-related infections. This review undertakes a comprehensive analysis of the literature from a historic perspective commenting on the contribution of the different models and discussing future venues and new approaches that can be merged with more traditional techniques in order to model biofilm-infections and efficiently fight them. PMID:25437038

  1. Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients

    PubMed Central

    Al-Aloul, M; Crawley, J; Winstanley, C; Hart, C; Ledson, M; Walshaw, M

    2004-01-01

    Background: Chronic pulmonary infection with transmissible Pseudomonas aeruginosa strains in individuals with cystic fibrosis (CF) has been reported, raising issues of cross infection and patient segregation. The first such strain to be described (the Liverpool epidemic strain, LES) is now widespread in many UK CF centres. However, whether such infection carries a worse prognosis is unknown. To address this, the clinical course of a group of CF patients chronically infected by LES was compared with that in patients harbouring unique strains. Methods: Using P aeruginosa strain genotyping, two cohorts of CF patients attending the Liverpool CF service were identified who were LES positive or negative in 1998 and remained so until 2002. From these, two groups of 12 patients were matched in 1998 for age, spirometric parameters, and nutritional state and their clinical course was followed for 5 years. Patients chronically infected with Burkholderia cepacia were excluded. Results: Patients chronically infected with LES had a greater annual loss of lung function than those not chronically infected by LES (mean difference between groups -4.4% (95% CI -8.1 to -0.9; p<0.02)), and by 2002 their percentage predicted forced expiratory volume in 1 second (FEV1) was worse (mean 65.0% v 82.6%, p<0.03). Their nutritional state also deteriorated over the study period (mean difference between groups in body mass index -0.7 (95% CI -1.2 to -0.2; p<0.01)), such that by 2002 they were malnourished compared with LES negative patients (mean BMI 19.4 v 22.7, p<0.02). Conclusions: Chronic infection with the Liverpool epidemic P aeruginosa strain in CF patients confers a worse prognosis than infection with unique strains alone, confirming the need for patient segregation. Since this strain is common in many CF units, strain identification in all CF centres is essential. This can only be carried out using genomic typing methods. PMID:15047956

  2. Frequency of Pseudomonas aeruginosa serotypes in burn wound infections and their resistance to antibiotics.

    PubMed

    Estahbanati, Hamid Karimi; Kashani, Parnian Pour; Ghanaatpisheh, Fahimeh

    2002-06-01

    Pseudomonas aeruginosa plays a prominent role as an etiological agent involved in serious infections in burned patients. In this study P. aeruginosa infections were analyzed at the Motahari Burn Center in Tehran (from 22 December 1998 to April 1999) to estimate their frequency, antibiotic susceptibility and serotypes. One hundred and eighty-four positive cultures and 205 bacterial strains were isolated among swabs or biopsy specimens during the study period. Pseudomonas was found to be the most common (57%) followed by Acinetobacter (17%), Escherichia coli (12%), Staphylococcus aureus (8%) and other organisms (6%). The frequency of P. aeruginosa resistance to gentamicin, ceftizoxime, carbenicillin, cephalothin and ceftazidime was over 90%. The antibiotics to which P. aeruginosa was most sensitive were amikacin and tetracyclin. The "O" serotypes isolated from the 117 Pseudomona aeroginosa isolates were serotypes O:2, O:5, O:6, O:8, O:11, O:12 and O:16. The most common serotype was O:6 (20/17%) followed by O:11 (18/15%) and O:5 (14/12%). The serotype most resistant was O:16 (8%) and the most sensitive was O:8 (2%). Since treatment of infection with available antibiotics according to the results attained proved to be difficult, prevention of infection in the burned patients is considered as an appropriate means of conquering overcoming infection problems. The sum of frequencies of serotypes O:6, O:11, O:5 and O:16 was more than 60%, therefore vaccination of burn patients with polyvalent antiserum to these serotypes could possibly produce immunity in more than half of the burned patients. PMID:12052372

  3. Antiseptics for treating infected wounds: Efficacy on biofilms and effect of pH.

    PubMed

    Percival, Steven L; Finnegan, Simon; Donelli, Gianfranco; Vuotto, Claudia; Rimmer, Steve; Lipsky, Benjamin A

    2016-01-01

    Biofilm recalcitrance is a persistent problem when managing difficult to heal and infected chronic wounds. The wound biofilm is a fundamental factor in the re-occurrence and delayed healing commonly observed in non-healing and infected chronic wounds. However, there is presently no single antimicrobial agent that is completely efficacious against both the planktonic and sessile polymicrobial communities evident in at risk or infected wounds. We will review currently available antimicrobials, with particular emphasis on silver and iodine, employed to help suppress biofilms in wounds. In addition, we will also review the effect of pH on antimicrobial efficacy. Available evidence suggests that it is best to take a multifactorial approach towards controlling biofilm in chronic, "at risk" and infected wounds. This highlights the growing importance of avoiding indiscriminate or inappropriate use of antimicrobials in the treatment of chronic wounds. PMID:25159044

  4. Bacteriophage-based therapy in cystic fibrosis-associated Pseudomonas aeruginosa infections: rationale and current status

    PubMed Central

    Hraiech, Sami; Brégeon, Fabienne; Rolain, Jean-Marc

    2015-01-01

    Pulmonary infections involving Pseudomonas aeruginosa are among the leading causes of the deterioration of the respiratory status of cystic fibrosis (CF) patients. The emergence of multidrug-resistant strains in such populations, favored by iterative antibiotic cures, has led to the urgent need for new therapies. Among them, bacteriophage-based therapies deserve a focus. One century of empiric use in the ex-USSR countries suggests that bacteriophages may have beneficial effects against a large range of bacterial infections. Interest in bacteriophages has recently renewed in Western countries, and the in vitro data available suggest that bacteriophage-based therapy may be of significant interest for the treatment of pulmonary infections in CF patients. Although the clinical data concerning this specific population are relatively scarce, the beginning of the first large randomized study evaluating bacteriophage-based therapy in burn infections suggests that the time has come to assess the effectiveness of this new therapy in CF P. aeruginosa pneumonia. Consequently, the aim of this review is, after a brief history, to summarize the evidence concerning bacteriophage efficacy against P. aeruginosa and, more specifically, the in vitro studies, animal models, and clinical trials targeting CF. PMID:26213462

  5. Les infections à Pseudomonas aeruginosa au service des maladies infectieuses du CHU YO, Burkina Faso: à propos deux cas

    PubMed Central

    Mamoudou, Savadogo; Lassina, Dao; Fla, Koueta

    2015-01-01

    Nous rapportons deux cas d'infection à Pseudomonas aeruginosa: un cas de méningite et un cas d'infection urinaire. Les auteurs rappellent qu’à côté des étiologies classiques des méningites et des infections urinaires, des germes résistants comme Pseudomonas aeruginosa peuvent être responsables d'infections à localisation méningées et urinaires et dont il faut connaître pour une bonne prise en charge. Le traitement de ces infections requiert un antibiogramme au regard de la grande capacité de résistance de Pseudomonas aeruginosa en milieu hospitalier. La limitation des gestes invasifs et l'application rigoureuse des mesures de prévention des infections en milieu hospitalier contribueront à lutter efficacement contre ces infections en milieu de soins. PMID:26491521

  6. [Pathophysiology of implant-associated infections: From biofilm to osteolysis and septic loosening].

    PubMed

    Wagner, C; Hänsch, G M

    2015-12-01

    Biofilm formation is the key factor in the pathogenesis of implant-associated infections. The most common pathogens isolated are Staphylococcus species, opportunists belonging to the physiological flora of the skin. Biofilm formation starts with the adhesion of bacteria and colonisation preferentially occurs on the surfaces of the foreign body material. As an interactive symbiotic "city of microbes," biofilm formation represents an efficient survival strategy for bacteria. In clinically apparent infections the biofilm induces a local host response with infiltration of phagocytic immune cells. The proinflammatory microenvironment results in a stimulation of osteoclastogenesis, with local osteolysis, and finally septic loosening of the implant. According to the biofilm theory, retaining the implant in primary revision surgery is only recommended in early-stage infections with a stable implant; in late-stage infections, or when loosening occurs, the implant should be removed. Results of previous anti-biofilm therapies have not been satisfactory; therefore, current research is focused on prevention strategies, especially the modification of implant surfaces. Basic knowledge of the underlying pathophysiology is a prerequisite for the development of innovative interdisciplinary therapy and prevention strategies; in this context, essential aspects of biofilm formation, its consequences, and its relevance to diagnosis and therapy are described and discussed. PMID:26556489

  7. Photodynamic inactivation of biofilm: taking a lightly colored approach to stubborn infection

    PubMed Central

    de Melo, Wanessa CMA; Avci, Pinar; de Oliveira, Milene Nóbrega; Gupta, Asheesh; Vecchio, Daniela; Sadasivam, Magesh; Chandran, Rakkiyappan; Huang, Ying-Ying; Yin, Rui; Perussi, Livia R; Tegos, George P; Perussi, Janice R; Dai, Tianhong; Hamblin, Michael R

    2015-01-01

    Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents. PMID:23879608

  8. Photodynamic inactivation of biofilm: taking a lightly colored approach to stubborn infection.

    PubMed

    de Melo, Wanessa C M A; Avci, Pinar; de Oliveira, Milene Nóbrega; Gupta, Asheesh; Vecchio, Daniela; Sadasivam, Magesh; Chandran, Rakkiyappan; Huang, Ying-Ying; Yin, Rui; Perussi, Livia R; Tegos, George P; Perussi, Janice R; Dai, Tianhong; Hamblin, Michael R

    2013-07-01

    Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents. PMID:23879608

  9. Immunoproteomic Identification of In Vivo-Produced Propionibacterium acnes Proteins in a Rabbit Biofilm Infection Model

    PubMed Central

    Achermann, Yvonne; Tran, Bao; Kang, Misun; Harro, Janette M.

    2015-01-01

    Propionibacterium acnes is well-known as a human skin commensal but can also act as an invasive pathogen causing implant-associated infections. In order to resolve these types of P. acnes infections, the implants must be removed, due to the presence of an established biofilm that is recalcitrant to antibiotic therapy. In order to identify those P. acnes proteins produced in vivo during a biofilm infection, we established a rabbit model of implant-associated infection with this pathogen. P. acnes biofilms were anaerobically grown on dextran beads that were then inoculated into the left tibias of rabbits. At 4 weeks postinoculation, P. acnes infection was confirmed by radiograph, histology, culture, and PCR. In vivo-produced and immunogenic P. acnes proteins were detected on Western blot using serum samples from rabbits infected with P. acnes after these bacterial proteins were separated by two-dimensional gel electrophoresis. Those proteins that bound host antibodies were then isolated and identified by tandem mass spectrometry. Radiographs and histology demonstrated a disruption in the normal bone architecture and adherent biofilm communities in those animals with confirmed infections. A total of 24 immunogenic proteins were identified; 13 of these proteins were upregulated in both planktonic and biofilm modes, including an ABC transporter protein. We successfully adapted a rabbit model of implant-associated infection for P. acnes to identify P. acnes proteins produced during a chronic biofilm-mediated infection. Further studies are needed to evaluate the potential of these proteins for either a diagnostic test or a vaccine to prevent biofilm infections caused by P. acnes. PMID:25694647

  10. Flagellin induces myeloid-derived suppressor cells: implications for Pseudomonas aeruginosa infection in cystic fibrosis lung disease.

    PubMed

    Rieber, Nikolaus; Brand, Alina; Hector, Andreas; Graepler-Mainka, Ute; Ost, Michael; Schäfer, Iris; Wecker, Irene; Neri, Davide; Wirth, Andreas; Mays, Lauren; Zundel, Sabine; Fuchs, Jörg; Handgretinger, Rupert; Stern, Martin; Hogardt, Michael; Döring, Gerd; Riethmüller, Joachim; Kormann, Michael; Hartl, Dominik

    2013-02-01

    Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa-infected CF patient ex vivo-isolated as well as flagellin or P. aeruginosa in vitro-generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell-mediated host defense in CF and other P. aeruginosa-associated chronic lung diseases. PMID:23277486

  11. Novel bacteriophage therapy for controlling metallo-beta-lactamase producing Pseudomonas aeruginosa infection in Catfish

    PubMed Central

    2013-01-01

    Background The bacteriophage therapy is an effective antimicrobial approach with potentially important applications in medicine and biotechnology which can be seen as an additional string in the bow. Emerging drug resistant bacteria in aquaculture industry due to unrestricted use of antibiotics warrants more sustainable and environmental friendly strategies for controlling fish infections. The isolated bacteria from fish lesions was characterised based on isolation on selective and differential medium like Pseudomonas agar, gram staining, biochemical tests and 16SrRNA sequencing. The metallo-beta-lactamase (MBL) producing bacterial isolate was evaluated using Imipenem - Ethylenediaminetetraacetic acid (EDTA) disk method. The specific bacteriophage was isolated and concentrated using coal bed developed in our lab at CSIR-NEERI. The isolated and enriched bacteriophage was characterised by nucleotide sequencing and electron microscopy. The phage therapy was applied for treating ulcerative lesion in fish. Results The pathogenic bacterium responsible for causing ulcerative lesions in catfish species (Clarias gariepinus) was identified as Pseudomonas aeruginosa. One out of twenty P. aeruginosa isolate showing multi drug resistance (MDR) was incidentally found to be MBL producing as determined by Imipenem-EDTA disk method. The phage therapy effectively cured the ulcerative lesions of the infected fish in 8–10 days of treatment, with a sevenfold reduction of the lesion with untreated infection control. Conclusion Bacteriophage therapy can have potential applications soon as an alternative or as a complement to antibiotic treatment in the aquaculture. We present bacteriophage therapy as a treatment method for controlling MDR P. aeruginosa infection in C. gariepinus. To the best of our knowledge this is a first report of application of phage therapy against MBL producing P. aeruginosa isolated from aquatic ecosystem. PMID:24369750

  12. Prevention of bloodstream infections by photodynamic inactivation of multiresistant Pseudomonas aeruginosa in burn wounds

    NASA Astrophysics Data System (ADS)

    Hashimoto, M. C. E.; Prates, R. A.; Toffoli, D. J.; Courrol, L. C.; Ribeiro, M. S.

    2010-02-01

    Bloodstream infections are potentially life-threatening diseases. They can cause serious secondary infections, and may result in endocarditis, severe sepsis or toxic-shock syndrome. Pseudomonas aeruginosa is an opportunistic pathogen and one of the most important etiological factors responsible for nosocomial infections, mainly in immuno-compromissed hosts, characteristic of patients with severe burns. Its multiresistance to antibiotics produces many therapeutic problems, and for this reason, the development of an alternative method to antibiotic therapy is needed. Photodynamic inactivation (PDI) may be an effective and alternative therapeutic option to prevent bloodstream infections in patients with severe burns. In this study we report the use of PDI to prevent bloodstream infections in mice with third-degree burns. Burns were produced on the back of the animals and they were infected with 109 cfu/mL of multi-resistant (MR) P. aeruginosa. Fifteen animals were divided into 3 groups: control, PDT blue and PDT red. PDT was performed thirty minutes after bacterial inoculation using 10μM HB:La+3 and a light-emitting diode (LED) emitting at λ=460nm+/-20nm and a LED emitting at λ=645 nm+/-10nm for 120s. Blood of mice were colected at 7h, 10h, 15h, 18h and 22h pos-infection (p.i.) for bacterial counting. Control group presented 1×104 cfu/mL in bloodstream at 7h p.i. increasing to 1×106 at 22h, while mice PDT-treated did not present any bacteria at 7h; only at 22h p.i. they presented 1×104cfu/mL. These results suggest that HB:La+3 associated to blue LED or red LED is effective to delay and diminish MR P.aeruginosa bloodstream invasion in third-degree-burned mice.

  13. Study on antimicrobial potential of neem oil nanoemulsion against Pseudomonas aeruginosa infection in Labeo rohita.

    PubMed

    Mishra, Prabhakar; R S, Suresh Kumar; Jerobin, Jayakumar; Thomas, John; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2014-01-01

    Presence of several biochemical constituents in neem makes it an efficient antimicrobial agent for pathogenic diseases. The current investigation was aimed to assess the therapeutic potential of neem nanoemulsion as a control measure for Pseudomonas aeruginosa infection in freshwater fish Labeo rohita. The median lethal concentration (LC50) for the neem oil and neem nanoemulsion was 73.9 and 160.3 mg/L, respectively. The biomarker enzymes of treated fish tissues showed a significant difference in the level of glutathione reductase, catalase, and lipid peroxidation in neem oil-treated samples than in neem nanoemulsion-treated samples at P<0.05. The results were corroborative with histopathology and ultrastructural analysis. The bacterial infection of P. aeruginosa treated using neem nanoemulsion was more effective in both in vitro and in vivo methods. Present findings suggest that neem-based nanoemulsion has negligible toxicity to Rohu fishes. This makes neem-based nanoemulsion as an efficient therapeutic agent against P. aeruginosa infection, leading to its possible usage in the aquaculture industry. PMID:24502533

  14. Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites.

    PubMed

    Lassek, Christian; Berger, Antje; Zühlke, Daniela; Wittmann, Christoph; Riedel, Katharina

    2016-05-01

    Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel-free and data-independent LC-IMS(E) approach. Moreover, the metabolic diversity of these isolates was investigated by (13) C-metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified for the reference strain P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all isolates. Whilst the majority of these proteins were expressed in constant amounts, expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in burned wounds isolates compared to lung/urinary tract isolates. Moreover, proteins involved in iron/amino acids uptake were found to be highly abundant in urinary tract isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated proteome/fluxome analysis did not indicate straightforward correlation between the protein amount and flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments. All MS data have been deposited in the ProteomeXchange with identifier PXD002373 (http://proteomecentral.proteomexchange.org/dataset/PXD002373). PMID:26959854

  15. Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

    PubMed Central

    Vogeleer, Philippe; Tremblay, Yannick D. N.; Jubelin, Grégory; Jacques, Mario

    2015-01-01

    Forming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilm-associated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment. PMID:26712549

  16. Successful implementation of infection control strategies prevents P. aeruginosa transmission among cystic fibrosis patients inside the hospital

    PubMed Central

    Matt, Benedikt; Mitteregger, Dieter; Renner, Sabine; Presterl, Elisabeth; Assadian, Ojan; Diab-Elschahawi, Magda

    2014-01-01

    Background: The aim of this study was to characterise the epidemiology of P. aeruginosa isolated from cystic fibrosis (CF) patients at the Vienna General Hospital (VGH) by molecular genetic fingerprinting in order to understand transmission ways and to evaluate the established infection control protocols. Methods: The outpatient clinic for CF patients at the VGH cares for children and adolescents up to the age of 18 years. Among an average of 139 patients cared for at the clinic, 41 were tested positive for P. aeruginosa during the study period. Fifty P. aeruginosa isolates, obtained between August 2010 and March 2012 from routine examinations of CF patients, were subject to molecular characterization using the DiversiLab® method. Results: 42 distinguishable molecular-biological patterns were identified, 7 of which were found multiple times. 40 out of 42 genotypes were retrieved from single patients only, while two patterns were present in two patients each. Nine patients presented with two or more phenotypically diverse P. aeruginosa isolates. In five of these cases the retrieved isolates belonged to the same genotype. Conclusion: The broad genetic heterogeneity of P. aeruginosa in the studied patient population suggests that the majority of CF patients cared for at the VGH acquire P. aeruginosa from environmental sources. It may be concluded that implemented infection control guidelines have been successful in preventing nosocomial transmission of P. aeruginosa among CF patients within the VGH and patient-to-patient transmission outside the hospital. Chronic polyclonal infection/colonization was rare in the study population. PMID:25285264

  17. Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

    PubMed

    Scoffield, Jessica A; Wu, Hui

    2016-02-01

    Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis. PMID:26673783

  18. Bacterial Biofilms on Implanted Suture Material Are a Cause of Surgical Site Infection

    PubMed Central

    Nistico, Laura; Tower, Irene; Lasko, Leslie-Ann; Stoodley, Paul

    2014-01-01

    Abstract Background: Surgical site infection (SSI) has been estimated to occur in up to 5% of all procedures, accounting for up to 0.5% of all hospital costs. Bacterial biofilms residing on implanted foreign bodies have been implicated as contributing or causative factors in a wide variety of infectious scenarios, but little consideration has been given to the potential for implanted, submerged suture material to act as a host for biofilm and thus serve as a nidus of infection. Methods: We report a series of 15 patients who underwent open Roux-en-Y gastric bypass (with musculofascial closure with permanent, multifilament sutures) who developed longstanding and refractory SSIs in the abdominal wall. Explanted suture material at subsequent exploration was examined for biofilm with confocal laser-scanning microscopy (CLSM) and fluorescence in situ hybridization (FISH). Results: All 15 patients at re-exploration were found to have gross evidence of a “slimy” matrix or dense reactive granulation tissue localized to the implanted sutures. Confocal laser-scanning microscopy revealed abundant biofilm present on all sutures examined; FISH was able to identify the presence of specific pathogens in the biofilm. Complete removal of the foreign bodies (and attendant biofilms) resulted in all cases in cure of the SSI. Conclusion: Bacterial biofilms on implanted suture material can manifest as persistent surgical site infections that require complete removal of the underlying foreign body substrata for resolution. PMID:24833403

  19. The Healing Effect of Licorice on Pseudomonas aeruginosa Infected Burn Wounds in Experimental Rat Model

    PubMed Central

    Tanideh, Nader; Rokhsari, Pedram; Mehrabani, Davood; Mohammadi Samani, Soleiman; Sabet Sarvestani, Fatemeh; Ashraf, Mohammad Javad; Koohi Hosseinabadi, Omid; Shamsian, Shahram; Ahmadi, Nasrollah

    2014-01-01

    BACKGROUND Burn is still one of the most devastating injuries in emergency medicine while improvements in wound healing knowledge and technology have resulted into development of new dressings. This study was undertaken to evaluate the healing effect of licorice in Pseudomonas aeruginosa infected burn wounds of experimental rat model. METHODS One hundred and twenty female Sprague-Dawley rats were randomly allocated to 4 equal groups. Group A received silver sulfadiazine ointment, Group B received 10% licorice extract and Group C was considered as control group and received gel base as the base of medication. Group D did not receive any medication and just underwent burn injury. A standard 3rd degree burn wound was produced by a hot plate with similar size about 20% of total body surface area (TBSA) and at identical temperature. After 24 h of burn production, 108 colony forming units (CFU) of toxigenic strains of P. aeruginosa (PA 103) were inoculated subcutaneously into the burnt area. After 3, 7, 14, 21 and 28 days of therapy, the animals were sacrificed and burn areas were macroscopically examined and histologically evaluated. RESULTS Decrease in size of the burn wounds, in inflammation and re-epithelialization were poor in groups B-D. Infection to P. aeruginosa was still visible in groups B-D but was absent in Group A. The mean histological score, tensile strength, maximum stress, yield strength and stiffness in groups B-D were lower compared with Group A. CONCLUSION Licorice extract in 10% concentration was shown not to be effective in healing of P. aeruginosa infected burn wounds. PMID:25489532

  20. The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction

    PubMed Central

    Wakeman, Catherine A.; Moore, Jessica L.; Noto, Michael J.; Zhang, Yaofang; Singleton, Marc D.; Prentice, Boone M.; Gilston, Benjamin A.; Doster, Ryan S.; Gaddy, Jennifer A.; Chazin, Walter J.; Caprioli, Richard M.; Skaar, Eric P.

    2016-01-01

    Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections. PMID:27301800

  1. The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction.

    PubMed

    Wakeman, Catherine A; Moore, Jessica L; Noto, Michael J; Zhang, Yaofang; Singleton, Marc D; Prentice, Boone M; Gilston, Benjamin A; Doster, Ryan S; Gaddy, Jennifer A; Chazin, Walter J; Caprioli, Richard M; Skaar, Eric P

    2016-01-01

    Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections. PMID:27301800

  2. Specific Resistance to Pseudomonas aeruginosa Infection in Zebrafish Is Mediated by the Cystic Fibrosis Transmembrane Conductance Regulator ▿ †

    PubMed Central

    Phennicie, Ryan T.; Sullivan, Matthew J.; Singer, John T.; Yoder, Jeffrey A.; Kim, Carol H.

    2010-01-01

    Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ΔLasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis. PMID:20732993

  3. Biofilms: An Underappreciated Mechanism of Treatment Failure and Recurrence in Vaginal Infections.

    PubMed

    Muzny, Christina A; Schwebke, Jane R

    2015-08-15

    Biofilms are microbial communities of surface-attached cells embedded in a self-produced extracellular matrix. They are of major medical significance because they decrease susceptibility to antimicrobial agents and enhance the spread of antimicrobial resistance. Biofilm-associated bacterial and fungal microorganisms have increasingly been recognized to play a role in multiple infectious diseases, particularly in their persistence and recurrence. More recently, biofilms have also been implicated in vaginal infections, notably bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), particularly in the setting of treatment failure and recurrence. The purpose of this review is to discuss the impact of biofilms on the management and treatment of BV and recurrent VVC and highlight the need for additional research and development of novel therapeutics targeting pathogenic vaginal biofilms. PMID:25935553

  4. Biofilms: An Underappreciated Mechanism of Treatment Failure and Recurrence in Vaginal Infections

    PubMed Central

    Muzny, Christina A.; Schwebke, Jane R.

    2015-01-01

    Biofilms are microbial communities of surface-attached cells embedded in a self-produced extracellular matrix. They are of major medical significance because they decrease susceptibility to antimicrobial agents and enhance the spread of antimicrobial resistance. Biofilm-associated bacterial and fungal microorganisms have increasingly been recognized to play a role in multiple infectious diseases, particularly in their persistence and recurrence. More recently, biofilms have also been implicated in vaginal infections, notably bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), particularly in the setting of treatment failure and recurrence. The purpose of this review is to discuss the impact of biofilms on the management and treatment of BV and recurrent VVC and highlight the need for additional research and development of novel therapeutics targeting pathogenic vaginal biofilms. PMID:25935553

  5. Role of Nonagglutinating Antibody in the Protracted Immunity of Vaccinated Mice to Pseudomonas aeruginosa Infection

    PubMed Central

    Moody, M. R.; Kessel, R. W. I.; Young, V. M.; Fiset, P.

    1978-01-01

    Effective immunization against infection with Pseudomonas aeruginosa is difficult to evaluate because agglutinin levels decline rapidly. Because fractionation of hyperimmune sera often yields more specific antibody than can be accounted for by direct agglutination tests, an immunoglobulin-specific assay based on antiglobulin augmentation was used to characterize antibody responses of C3H/HeJ mice vaccinated with P. aeruginosa type 2 lipopolysaccharide. Nonagglutinating antibodies, initially detected at 2 weeks post-primary vaccination, were predominantly immunoglobulin G after 5 weeks, and they remained elevated at levels usually 32-fold higher than the direct titer throughout the 4-month study period. The sequential production of immunoglobulin M, then immunoglobulin G, followed that found in orthodox immunological responses. Sera that contained nonagglutinating antibodies but not direct agglutinins (14 to 16 weeks) enhanced phagocytosis of P. aeruginosa type 2 by macrophages from unimmunized mice and passively immunized mice against lethal challenge doses; bactericidal activity of these sera was not demonstrated in the presence or absence of complement. When challenged with 1, 10, and 100 50% lethal doses at 16 weeks, survival rates of actively immunized mice were significantly higher than those of unvaccinated mice (P < 0.001). Thus, at a time when no direct agglutinins were detectable, the augmented system detected nonagglutinating antibodies that could confer protracted resistance in vaccinated mice to pseudomonas infection. PMID:101467

  6. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

    PubMed Central

    Bohn, Yu-Sing Tammy; Brandes, Gudrun; Rakhimova, Elza; Horatzek, Sonja; Salunkhe, Prabhakar; Munder, Antje; van Barneveld, Andrea; Jordan, Doris; Bredenbruch, Florian; Häußler, Susanne; Riedel, Kathrin; Eberl, Leo; Jensen, Peter Østrup; Bjarnsholt, Thomas; Moser, Claus; Hoiby, Niels; Tümmler, Burkhard; Wiehlmann, Lutz

    2008-01-01

    Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population. PMID:19054330

  7. The Healing Effect of Scrophularia Striata on Experimental Burn Wounds Infected to Pseudomonas Aeruginosa in Rat

    PubMed Central

    Tanideh, Nader; Haddadi, Mohammad Hossein; Rokni-Hosseini, Mohammad Hossein; Hossienzadeh, Masood; Mehrabani, Davood; Sayehmiri, Kourosh; Koohi-Hossienabadi, Omid

    2015-01-01

    BACKGROUND The cause of death in burn patients after 48 hours of hospitalization has been reported to be bacterial infections. Recently, due to the compounds accelerating the healing process and the intense reduction of treatment side effects, medicinal plants are used to cure burn wound infections. This study aims to investigate the medicinal effect of the ethanolic extract of Scrophularia striata on burn wound infection in in-vivo and in-vitro in comparison with silver sulfadiazine (SSD). METHODS One hundred and fifty male Sprague Dawley rats were divided into 3 equal groups. A hot plate of 1×1cm was used to create second degree burn wounds. The ethanolic extract of S. striata was provided through percolation method. Group 1 was treated with SSD, group 2 with S. striata, and group 3 was considered as control group. All animals were infected to Pseudomonas aeruginosa. On days 3, 7, 10, 14, and 21 after burn wound injury, the animals were euthanized and were evaluated histologically. The MIC and MBC were determined using the micro dilution method. RESULTS The rate of wound healing was significantly greater in S. striata group in comparison to SSD and control groups. CONCLUSION S. striata contains was shown to have anti-bacterial and wound healing effects while this effect was significantly more than SSD denoting to its use when needed for burn wounds infected to P. aeruginosa. PMID:25606472

  8. Biofilms and persistent wound infections in United States military trauma patients: a case–control analysis

    PubMed Central

    2014-01-01

    Background Complex traumatic injuries sustained by military personnel, particularly when involving extremities, often result in infectious complications and substantial morbidity. One factor that may further impair patient recovery is the persistence of infections. Surface-attached microbial communities, known as biofilms, may play a role in hindering the management of infections; however, clinical data associating biofilm formation with persistent or chronic infections are lacking. Therefore, we evaluated the production of bacterial biofilms as a potential risk factor for persistent infections among wounded military personnel. Methods Bacterial isolates and clinical data from military personnel with deployment-related injuries were collected through the Trauma Infectious Disease Outcomes Study. The study population consisted of patients with diagnosed skin and soft-tissue infections. Cases (wounds with bacterial isolates of the same organism collected 14 days apart) were compared to controls (wounds with non-recurrent bacterial isolates), which were matched by organism and infectious disease syndrome. Potential risk factors for persistent infections, including biofilm formation, were examined in a univariate analysis. Data are expressed as odds ratios (OR; 95% confidence interval [CI]). Results On a per infected wound basis, 35 cases (representing 25 patients) and 69 controls (representing 60 patients) were identified. Eight patients with multiple wounds were utilized as both cases and controls. Overall, 235 bacterial isolates were tested for biofilm formation in the case–control analysis. Biofilm formation was significantly associated with infection persistence (OR: 29.49; CI: 6.24-infinity) in a univariate analysis. Multidrug resistance (OR: 5.62; CI: 1.02-56.92), packed red blood cell transfusion requirements within the first 24 hours (OR: 1.02; CI: 1.01-1.04), operating room visits prior to and on the date of infection diagnosis (OR: 2.05; CI: 1

  9. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

    PubMed

    Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon; Nile, Christopher

    2015-08-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections. PMID:26092919

  10. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

    PubMed Central

    Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F.; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon

    2015-01-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections. PMID:26092919

  11. Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics

    PubMed Central

    Lebeaux, David; Ghigo, Jean-Marc

    2014-01-01

    SUMMARY Surface-associated microbial communities, called biofilms, are present in all environments. Although biofilms play an important positive role in a variety of ecosystems, they also have many negative effects, including biofilm-related infections in medical settings. The ability of pathogenic biofilms to survive in the presence of high concentrations of antibiotics is called “recalcitrance” and is a characteristic property of the biofilm lifestyle, leading to treatment failure and infection recurrence. This review presents our current understanding of the molecular mechanisms of biofilm recalcitrance toward antibiotics and describes how recent progress has improved our capacity to design original and efficient strategies to prevent or eradicate biofilm-related infections. PMID:25184564

  12. Exploring Dangerous Connections between Klebsiella pneumoniae Biofilms and Healthcare-Associated Infections

    PubMed Central

    Bandeira, Maria; Almeida Carvalho, Patricia; Duarte, Aida; Jordao, Luisa

    2014-01-01

    Healthcare-associated infections (HAI) are a huge public health concern, particularly when the etiological agents are multidrug resistant. The ability of bacteria to develop biofilm is a helpful skill, both to persist within hospital units and to increase antibiotic resistance. Although the links between antibiotic resistance, biofilms assembly and HAI are consensual, little is known about biofilms. Here, electron microscopy was adopted as a tool to investigate biofilm structures associated with increased antibiotic resistance. The K. pneumoniae strains investigated are able to assemble biofilms, albeit with different kinetics. The biofilm structure and the relative area fractions of bacteria and extracellular matrix depend on the particular strain, as well as the minimal inhibitory concentration (MIC) for the antibiotics. Increased values were found for bacteria organized in biofilms when compared to the respective planktonic forms, except for isolates Kp45 and Kp2948, the MIC values for which remained unchanged for fosfomycin. Altogether, these results showed that the emergence of antimicrobial resistance among bacteria responsible for HAI is a multifactorial phenomenon dependent on antibiotics and on bacteria/biofilm features. PMID:25438020

  13. Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

    PubMed Central

    Rakhimova, Elza; Munder, Antje; Wiehlmann, Lutz; Bredenbruch, Florian; Tümmler, Burkhard

    2008-01-01

    Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild. PMID:18301762

  14. Next Generation Sequencing Analysis of Biofilms from Three Dogs with Postoperative Surgical Site Infection

    PubMed Central

    König, L. M.; Klopfleisch, R.; Höper, D.; Gruber, A. D.

    2014-01-01

    The composition of biofilms in chronic wound infections of dogs is unclear. In the present study, histologically identified biofilms attached to sutures in chronically infected wounds of three dogs were examined by next generation sequencing of total DNA extracted from formalin-fixed and paraffin-embedded tissue samples. The analysis identified an inhomogeneous bacterial composition in three tissues containing biofilms. Some of the identified bacterial families such as Staphylococci and Streptococci have been found before in biofilms associated with human and canine wounds but in this study were quantitatively in the minority. The majority of the reads classified as bacterial sequences had the highest identity with sequences belonging to the Porphyromonadaceae, Deinococcaceae, Methylococcaceae, Nocardiaceae, Alteromonadaceae, and Propionibacteriaceae and thus taxons of so far minor relevance in veterinary medicine. PMID:27355023

  15. Proteomic profiling of host-biofilm interactions in an oral infection model resembling the periodontal pocket

    PubMed Central

    Bao, Kai; Belibasakis, Georgios N.; Selevsek, Nathalie; Grossmann, Jonas; Bostanci, Nagihan

    2015-01-01

    Periodontal infections cause inflammatory destruction of the tooth supporting tissues. We recently developed a dynamic, in vitro periodontal organotypic tissue model in a perfusion bioreactor system, in co-culture with an 11-species subgingival biofilm, which may recapitulate early events during the establishment of periodontal infections. This study aimed to characterize the global proteome regulations in this host-biofilm interaction model. Semi-quantitative shotgun proteomics were applied for protein identification and quantification in the co-culture supernatants (human and bacterial) and the biofilm lysates (bacterial). A total of 896 and 3363 proteins were identified as secreted in the supernatant and expressed in the biofilm lysate, respectively. Enriched gene ontology analysis revealed that the regulated secreted human tissue proteins were related to processes of cytoskeletal rearrangement, stress responses, apoptosis, and antigen presentation, all of which are commensurate with deregulated host responses. Most secreted bacterial biofilm proteins derived from their cytoplasmic domain. In the presence of the tissue, the levels of Fusobacterium nucleatum, Actinomyces oris and Campylobacter rectus proteins were significantly regulated. The functions of the up-regulated intracellular (biofilm lysate) proteins were associated with cytokinesis. In conclusion, the proteomic overview of regulated pathways in this host-biofilm interaction model provides insights to the early events of periodontal pathogenesis. PMID:26525412

  16. The ferrichrome receptor A as a new target for Pseudomonas aeruginosa virulence attenuation.

    PubMed

    Lee, Keehoon; Lee, Kang-Mu; Go, Junhyeok; Ryu, Jae-Chan; Ryu, Ji-Hwan; Yoon, Sang Sun

    2016-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen. PMID:27190289

  17. Pseudomonas aeruginosa RhlR is required to neutralize the cellular immune response in a Drosophila melanogaster oral infection model

    PubMed Central

    Limmer, Stefanie; Haller, Samantha; Drenkard, Eliana; Lee, Janice; Yu, Shen; Kocks, Christine; Ausubel, Frederick M.; Ferrandon, Dominique

    2011-01-01

    An in-depth mechanistic understanding of microbial infection necessitates a molecular dissection of host–pathogen relationships. Both Drosophila melanogaster and Pseudomonas aeruginosa have been intensively studied. Here, we analyze the infection of D. melanogaster by P. aeruginosa by using mutants in both host and pathogen. We show that orally ingested P. aeruginosa crosses the intestinal barrier and then proliferates in the hemolymph, thereby causing the infected flies to die of bacteremia. Host defenses against ingested P. aeruginosa included an immune deficiency (IMD) response in the intestinal epithelium, systemic Toll and IMD pathway responses, and a cellular immune response controlling bacteria in the hemocoel. Although the observed cellular and intestinal immune responses appeared to act throughout the course of the infection, there was a late onset of the systemic IMD and Toll responses. In this oral infection model, P. aeruginosa PA14 did not require its type III secretion system or other well-studied virulence factors such as the two-component response regulator GacA or the protease AprA for virulence. In contrast, the quorum-sensing transcription factor RhlR, but surprisingly not LasR, played a key role in counteracting the cellular immune response against PA14, possibly at an early stage when only a few bacteria are present in the hemocoel. These results illustrate the power of studying infection from the dual perspective of host and pathogen by revealing that RhlR plays a more complex role during pathogenesis than previously appreciated. PMID:21987808

  18. The application of biofilm science to the study and control of chronic bacterial infections

    PubMed Central

    Costerton, William; Veeh, Richard; Shirtliff, Mark; Pasmore, Mark; Post, Christopher; Ehrlich, Garth

    2003-01-01

    Unequivocal direct observations have established that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms. The diagnostic and therapeutic strategies that have served us so well in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases. We discuss the potential benefits of the application of the new methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases. PMID:14617746

  19. Host Genetic Background Influences the Response to the Opportunistic Pseudomonas aeruginosa Infection Altering Cell-Mediated Immunity and Bacterial Replication

    PubMed Central

    Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A.; Bragonzi, Alessandra

    2014-01-01

    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  20. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    PubMed

    De Simone, Maura; Spagnuolo, Lorenza; Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A; Bragonzi, Alessandra

    2014-01-01

    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  1. Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes

    PubMed Central

    Heussler, Gary E.; Cady, Kyle C.; Koeppen, Katja; Bhuju, Sabin; Stanton, Bruce A.

    2015-01-01

    ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. PMID:25968642

  2. The Effect of Infection Control Nurses on the Occurrence of Pseudomonas aeruginosa Healthcare-Acquired Infection and Multidrug-Resistant Strains in Critically-Ill Children

    PubMed Central

    Xu, Wei; He, Linxi; Liu, Chunfeng; Rong, Jian; Shi, Yongyan; Song, Wenliang; Zhang, Tao; Wang, Lijie

    2015-01-01

    Background Healthcare-acquired Pseudomonas aeruginosa (P. aeruginosa) infections in the Pediatric Intensive Care Unit (PICU), which have a high incidence, increase treatment costs and mortality, and seriously threaten the safety of critically ill children. It is essential to seek convenient and effective methods to control and prevent healthcare-acquired infections (HAIs). This research was conducted to study the effect of infection control nurses on the occurrence of P. aeruginosa HAIs and multi-drug resistance (MDR) strains in PICU. Methods The clinical data was divided into two groups, with the age ranging from 1 month to 14 years. One group of the critically ill patients(N = 3,722) was admitted to PICU from 2007 to 2010, without the management of infection control nurses. The other group of the critically ill patients (N = 3,943) was admitted to PICU from 2011 to 2013, with the management of infection control nurses. Compare the mortality, morbidity and the incidence of acquired P. aeruginosa infections to evaluate the effect of infection control nurses. Results After implementation of the post of infection control nurses, the patient's overall mortality fell from 4.81% to 3.73%. Among the patients with endotracheal intubation more than 48 hours, the incidence of endotracheal intubation-related pneumonia decreased from 44.6% to 34.32%. The mortality of patients with endotracheal intubation decreased from 16.96% to 10.17%, and the morbidity of HAIs with P. aeruginosa decreased from 1.89% to 1.07%. The mutual different rate (MDR) dropped from 67.95% to 44.23%. There were remarkable differences in these rates between the two groups (p<0.05). Conclusion Implementing the post of infection control nurses is associated with effectively reducing the HAI rate, especially the incidence and morbidity of P. aeruginosa HAIs, reducing PICU mortality, improving P. aeruginosa drug resistance. PMID:26630032

  3. The role of biofilm on orthopaedic implants: the "Holy Grail" of post-traumatic infection management?

    PubMed

    Mauffrey, C; Herbert, B; Young, H; Wilson, M L; Hake, M; Stahel, P F

    2016-08-01

    The development of post-traumatic infection is potentially a limb threatening condition. The orthopaedic trauma literature lags behind the research performed by our arthroplasty colleagues on the topic of implant-related infections. Surgical site infections in the setting of a recent ORIF are notoriously hard to eradicate due to biofilm formation around the implant. This bacteria-friendly, dynamic, living pluri-organism structure has the ability to morph and adapt to virtually any environment with the aim to maintain the causative organism alive. The challenges are twofold: establishing an accurate diagnosis with speciation/sensitivity and eradicating the infection. Multiple strategies have been researched to improve diagnostic accuracy, to prevent biofilm formation on orthopaedic implants, to mobilize/detach or weaken the biofilm or to target specifically bacteria embedded in the biofilm. The purpose of our paper is to review the patho-physiology of this mysterious pluri-cellular structure and to summarize some of the most pertinent research performed to improve diagnostic and treatment strategies in biofilm-related infections. PMID:27262848

  4. bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans

    PubMed Central

    Estes, Kathleen A.; Dunbar, Tiffany L.; Powell, Jennifer R.; Ausubel, Frederick M.; Troemel, Emily R.

    2010-01-01

    Very little is known about how animals discriminate pathogens from innocuous microbes. To address this question, we examined infection-response gene induction in the nematode Caenorhabditis elegans. We focused on genes that are induced in C. elegans by infection with the bacterial pathogen Pseudomonas aeruginosa, but are not induced by an isogenic attenuated gacA mutant. Most of these genes are induced independently of known immunity pathways. We generated a GFP reporter for one of these genes, infection response gene 1 (irg-1), which is induced strongly by wild-type P. aeruginosa strain PA14, but not by other C. elegans pathogens or by other wild-type P. aeruginosa strains that are weakly pathogenic to C. elegans. To identify components of the pathway that induces irg-1 in response to infection, we performed an RNA interference screen of C. elegans transcription factors. This screen identified zip-2, a bZIP transcription factor that is required for inducing irg-1, as well as several other genes, and is important for defense against infection by P. aeruginosa. These data indicate that zip-2 is part of a specialized pathogen response pathway that is induced by virulent strains of P. aeruginosa and provides defense against this pathogen. PMID:20133860

  5. Myeloid-derived suppressor cells (MDSCs) contribute to S. aureus orthopedic biofilm infection

    PubMed Central

    Heim, Cortney E.; Vidlak, Debbie; Scherr, Tyler D.; Kozel, Jessica A.; Holzapfel, Melissa; Muirhead, David E.; Kielian, Tammy

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes that are potent inhibitors of T cell activation. A role for MDSCs in bacterial infections has only recently emerged and nothing is known about MDSC function in the context of Staphylococcus aureus (S. aureus) infection. Since S. aureus biofilms are capable of subverting immune-mediated clearance, we examined whether MDSCs could play a role in this process. CD11b+Gr-1+ MDSCs represented the main cellular infiltrate during S. aureus orthopedic biofilm infection, accounting for over 75% of the CD45+ population. Biofilm-associated MDSCs inhibited T cell proliferation and cytokine production, which correlated with a paucity of T cell infiltrates at the infection site. Analysis of FACS-purified MDSCs recovered from S. aureus biofilms revealed increased Arg-1, iNOS, and IL-10 expression, key mediators of MDSC suppressive activity. Targeted depletion of MDSCs and neutrophils using the mAb 1A8 (anti-Ly6G) improved bacterial clearance by enhancing the intrinsic pro-inflammatory attributes of infiltrating monocytes and macrophages. Furthermore, the ability of monocytes/macrophages to promote biofilm clearance in the absence of MDSC action was revealed with RB6-C85 (anti-Gr-1 or anti-Ly6G/Ly6C) administration, which resulted in significantly increased S. aureus burdens both locally and in the periphery, since effector Ly-6C monocytes and by extension, mature macrophages, were also depleted. Collectively, these results are the first to demonstrate that MDSCs are key contributors to the chronicity of S. aureus biofilm infection, as their immunosuppressive function prevents monocyte/macrophage proinflammatory activity, which facilitates biofilm persistence. PMID:24646737

  6. Urinary catheter indwelling clinical pathogen biofilm formation, exopolysaccharide characterization and their growth influencing parameters

    PubMed Central

    Murugan, Kasi; Selvanayaki, Krishnasamy; Al-Sohaibani, Saleh

    2015-01-01

    Self-reproducing microbial biofilm community mainly involved in the contamination of indwelling medical devices including catheters play a vital role in nosocomial infections. The catheter-associated urinary tract infection (CA-UTI) causative Staphylococcus aureus, Enterobacter faecalis, and Pseudomonas aeruginosa were selectively isolated, their phenotypic as well as genotypic biofilm formation, production and monomeric sugar composition of EPS as well as sugar, salt, pH and temperature influence on their in vitro biofilm formation were determined. From 50 culture positive urinary catheters S. aureus (24%), P. aeruginosa (18%), E. faecalis (14%) and others (44%) were isolated. The performed assays revealed their varying biofilm forming ability. The isolated S. aureus ica, E. faecalis esp, and P. aeruginosa cup A gene sequencing and phylogenetic analysis showed their close branching and genetic relationship. The analyzed sugar, salt, pH, and temperature showed that the degree of CA-UTI isolates biofilm formation is an environmentally sensitive process. EPS monosaccharide HPLC analysis showed the presence of neutral sugars (ng/μl) as follows: glucose (P. aeruginosa: 44.275; E. faecalis: 4.23), lactose (P. aeruginosa: 7.29), mannitol (P. aeruginosa: 2.53; S. aureus: 2.62; E. faecalis: 2.054) and maltose (E. faecalis: 7.0042) revealing species-specific presence and variation. This study may have potential clinical relevance for the easy diagnosis and management of CA-UTI. PMID:26858552

  7. Responses of Pseudomonas aeruginosa to antimicrobials

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2014-01-01

    Infections caused by Pseudomonas aeruginosa often are hard to treat; inappropriate chemotherapy readily selects multidrug-resistant P. aeruginosa. This organism can be exposed to a wide range of concentrations of antimicrobials during treatment; learning more about the responses of P. aeruginosa to antimicrobials is therefore important. We review here responses of the bacterium P. aeruginosa upon exposure to antimicrobials at levels below the inhibitory concentration. Carbapenems (e.g., imipenem) have been shown to induce the formation of thicker and more robust biofilms, while fluoroquinolones (e.g., ciprofloxacin) and aminoglycosides (e.g., tobramycin) have been shown to induce biofilm formation. Ciprofloxacin also has been demonstrated to enhance the frequency of mutation to carbapenem resistance. Conversely, although macrolides (e.g., azithromycin) typically are not effective against P. aeruginosa because of the pseudomonal outer-membrane impermeability and efflux, macrolides do lead to a reduction in virulence factor production. Similarly, tetracycline is not very effective against this organism, but is known to induce the type-III secretion system and consequently enhance cytotoxicity of P. aeruginosa in vivo. Of special note are the effects of antibacterials and disinfectants on pseudomonal efflux systems. Sub-inhibitory concentrations of protein synthesis inhibitors (aminoglycosides, tetracycline, chloramphenicol, etc.) induce the MexXY multidrug efflux system. This response is known to be mediated by interference with the translation of the leader peptide PA5471.1, with consequent effects on expression of the PA5471 gene product. Additionally, induction of the MexCD-OprJ multidrug efflux system is observed upon exposure to sub-inhibitory concentrations of disinfectants such as chlorhexidine and benzalkonium. This response is known to be dependent upon the AlgU stress response factor. Altogether, these biological responses of P. aeruginosa provide useful

  8. Responses of Pseudomonas aeruginosa to antimicrobials.

    PubMed

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2014-01-01

    Infections caused by Pseudomonas aeruginosa often are hard to treat; inappropriate chemotherapy readily selects multidrug-resistant P. aeruginosa. This organism can be exposed to a wide range of concentrations of antimicrobials during treatment; learning more about the responses of P. aeruginosa to antimicrobials is therefore important. We review here responses of the bacterium P. aeruginosa upon exposure to antimicrobials at levels below the inhibitory concentration. Carbapenems (e.g., imipenem) have been shown to induce the formation of thicker and more robust biofilms, while fluoroquinolones (e.g., ciprofloxacin) and aminoglycosides (e.g., tobramycin) have been shown to induce biofilm formation. Ciprofloxacin also has been demonstrated to enhance the frequency of mutation to carbapenem resistance. Conversely, although macrolides (e.g., azithromycin) typically are not effective against P. aeruginosa because of the pseudomonal outer-membrane impermeability and efflux, macrolides do lead to a reduction in virulence factor production. Similarly, tetracycline is not very effective against this organism, but is known to induce the type-III secretion system and consequently enhance cytotoxicity of P. aeruginosa in vivo. Of special note are the effects of antibacterials and disinfectants on pseudomonal efflux systems. Sub-inhibitory concentrations of protein synthesis inhibitors (aminoglycosides, tetracycline, chloramphenicol, etc.) induce the MexXY multidrug efflux system. This response is known to be mediated by interference with the translation of the leader peptide PA5471.1, with consequent effects on expression of the PA5471 gene product. Additionally, induction of the MexCD-OprJ multidrug efflux system is observed upon exposure to sub-inhibitory concentrations of disinfectants such as chlorhexidine and benzalkonium. This response is known to be dependent upon the AlgU stress response factor. Altogether, these biological responses of P. aeruginosa provide useful

  9. Protective effect of muramyl dipeptide analogs against infections of Pseudomonas aeruginosa or Candida albicans in mice.

    PubMed Central

    Fraser-Smith, E B; Matthews, T R

    1981-01-01

    Two analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) were found to give better protection than muramyl dipeptide against intraperitoneal Pseudomonas aeruginosa infection or intravenous Candida albicans infection in mice. The analogs tested were N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine and N-acetylmuramyl-L-alpha-amino-butyryl-D-isoglutamine. The optimum treatment was 80 mg/kg per day given once daily for 4 consecutive days before infection by the intraperitoneal, intravenous, or subcutaneous route. Dose response was limited. The compounds were not orally active. Synergism was seen between N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine and gentamicin. No postinfection protection was observed. A nonspecific stimulation of macrophage cells by muramyl dipeptide analogs may contribute to the protection because antiinfective activity against Listeria monocytogenes given intraperitoneally was achieved with CBA mice. PMID:7333666

  10. Characterization of corneal damage from Pseudomonas aeruginosa infection by the use of multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Chang, Yu-Lin; Chen, Wei-Liang; Lo, Wen; Chen, Shean-Jen; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2010-11-01

    Using multiphoton autofluorescence (MAF) and second harmonic generation (SHG) microscopy, we investigate the morphology and the structure of the corneal epithelium and stroma collagen of bovine cornea following injection of Pseudomonas aeruginosa. We found that corneal epithelial cells are damaged and stromal collagen becoming increasingly autofluorescent with time. We also characterized infected cornea cultured for 0, 6, 12, and 24 h by quantitative ratiometric MAF to SHG index (MAFSI) analysis. MAFSI results show that the destruction of the stromal collagen corresponds to a decrease in SHG intensity and increase of MAF signal with time.

  11. [A case of pulmonary infection caused by Pseudomonas aeruginosa with anorexia nervosa].

    PubMed

    Ugajin, Motoi; Miwa, Seiichi; Suda, Takafumi; Shirai, Masahiro; Hayakawa, Hiroshi; Chida, Kingo

    2009-12-01

    A 19-year-old woman with anorexia nervosa (body weight 25 kg) was admitted to our hospital, showing a cavitary shadow in the left upper lung field on a chest radiograph film. We diagnosed a pulmonary abscess caused by Pseudomonas aeruginosa. A few days later, exacerbation, including enlargement of the cavitary shadow, ipsilateral pleural effusion and bilateral infiltrations, were observed. Finally, by using antibiotics such as Doripenem (DRPM) and Ciprofloxacin (CPFX), we were able to treat the disease by bronchoscopic and thoracic drainage. This case suggests we should take carefully treat anorexia nervosa patients with pulmonary infection. PMID:20058691

  12. Interactions between Pseudomonas aeruginosa and Staphylococcus aureus during co-cultivations and polymicrobial infections.

    PubMed

    Nguyen, Angela T; Oglesby-Sherrouse, Amanda G

    2016-07-01

    Pseudomonas aeruginosa and Staphylococcus aureus are versatile bacterial pathogens and common etiological agents in polymicrobial infections. Microbial communities containing both of these pathogens are shaped by interactions ranging from parasitic to mutualistic, with the net impact of these interactions in many cases resulting in enhanced virulence. Polymicrobial communities of these organisms are further defined by multiple aspects of the host environment, with important implications for disease progression and therapeutic outcomes. This mini-review highlights the impact of these interactions on the host and individual pathogens, the molecular mechanisms that underlie these interactions, and host-specific factors that drive interactions between these two important pathogens. PMID:27236810

  13. Infectious Dose Dictates the Host Response during Staphylococcus aureus Orthopedic-Implant Biofilm Infection.

    PubMed

    Vidlak, Debbie; Kielian, Tammy

    2016-07-01

    Staphylococcus aureus is a leading cause of prosthetic joint infections (PJIs) that are typified by biofilm formation. Given the diversity of S. aureus strains and their propensity to cause community- or hospital-acquired infections, we investigated whether the immune response and biofilm growth during PJI were conserved among distinct S. aureus clinical isolates. Three S. aureus strains representing USA200 (UAMS-1), USA300 (LAC), and USA400 (MW2) lineages were equally effective at biofilm formation in a mouse model of PJI and elicited similar leukocyte infiltrates and cytokine/chemokine profiles. Another factor that may influence the course of PJI is infectious dose. In particular, higher bacterial inocula could accelerate biofilm formation and alter the immune response, making it difficult to discern underlying pathophysiological mechanisms. To address this issue, we compared the effects of two bacterial doses (10(3) or 10(5) CFU) on inflammatory responses in interleukin-12p40 (IL-12p40) knockout mice that were previously shown to have reduced myeloid-derived suppressor cell recruitment concomitant with bacterial clearance after low-dose challenge (10(3) CFU). Increasing the infectious dose of LAC to 10(5) CFU negated these differences in IL-12p40 knockout animals, demonstrating the importance of bacterial inoculum on infection outcome. Collectively, these observations highlight the importance of considering infectious dose when assessing immune responsiveness, whereas biofilm formation during PJI is conserved among clinical isolates commonly used in mouse S. aureus infection models. PMID:27091926

  14. In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus.

    PubMed

    Ferris, Ryan A; McCue, Patrick M; Borlee, Grace I; Loncar, Kristen D; Hennet, Margo L; Borlee, Bradley R

    2016-03-01

    In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent "biofilm-like" matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo. PMID:26719448

  15. In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

    PubMed Central

    McCue, Patrick M.; Borlee, Grace I.; Loncar, Kristen D.; Hennet, Margo L.

    2015-01-01

    In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent “biofilm-like” matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo. PMID:26719448

  16. BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of Pseudomonas aeruginosa

    PubMed Central

    Li, Yi; Petrova, Olga E.; Su, Shengchang; Lau, Gee W.; Panmanee, Warunya; Na, Renuka; Hassett, Daniel J.; Davies, David G.; Sauer, Karin

    2014-01-01

    The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing ΔbdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing ΔbdlA and ΔdipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung

  17. The clinical impact of bacterial biofilms

    PubMed Central

    Høiby, Niels; Ciofu, Oana; Johansen, Helle Krogh; Song, Zhi-jun; Moser, Claus; Jensen, Peter Østrup; Molin, Søren; Givskov, Michael; Tolker-Nielsen, Tim; Bjarnsholt, Thomas

    2011-01-01

    Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis. PMID:21485309

  18. Profile of Virulence Factors in the Multi-Drug Resistant Pseudomonas aeruginosa Strains of Human Urinary Tract Infections (UTI)

    PubMed Central

    Habibi, Asghar; Honarmand, Ramin

    2015-01-01

    Background: Putative virulence factors are responsible for the pathogenicity of UTIs caused by Pseudomonas aeruginosa (P. aeruginosa). Resistance of P. aeruginosa to commonly used antibiotics is caused by the extreme overprescription of those antibiotics. Objectives: The goal of the present study was to investigate the prevalence of virulence factors and the antibiotic resistance patterns of P. aeruginosa isolates in UTI cases in Iran. Patients and Methods: Two hundred and fifty urine samples were collected from patients who suffered from UTIs. Samples were cultured immediately, and those that were P. aeruginosa-positive were analyzed for the presence of virulence genes using polymerase chain reaction (PCR) testing. Antimicrobial susceptibility testing (AST) was performed using the disk diffusion method. Results: Of the 250 urine samples analyzed, 8 samples (3.2%) were positive for P. aeruginosa. The prevalence of P. aeruginosa in male and female patients was 2.7% and 3.5%, respectively, (P = 0.035). In patients less than 10 years old, it was 4.2%, and in patients more than 55 years old, it was 4.2%. These were the most commonly infected groups. The highest levels of resistance were seen against ampicillin (87.5%), norfloxacin (62.5%), gentamycin (62.5%), amikacin (62.5%), and aztreonam (62.5%), while the lowest were seen for meropenem (0%), imipenem (12.5%), and polymyxin B (12.5%). LasB (87.5%), pclH (75%), pilB (75%), and exoS (75%) were the most commonly detected virulence factors in the P. aeruginosa isolates. Conclusions: It is logical to first prescribe meropenem, imipenem, and polymyxin B in cases of UTIs caused by P. aeruginosa. Medical practitioners should be aware of the presence of levels of antibiotic resistance in hospitalized UTI patients in Iran. PMID:26756017