A c-di-GMP-Based Switch Controls Local Heterogeneity of Extracellular Matrix Synthesis which Is Crucial for Integrity and Morphogenesis of Escherichia coli Macrocolony Biofilms.
Serra Diego O,Hengge Regine
Journal of molecular biology
The extracellular matrix in macrocolony biofilms of Escherichia coli is arranged in a complex large-scale architecture, with homogenic matrix production close to the surface, whereas zones further below display pronounced local heterogeneity of matrix production, which results in distinct three-dimensional architectural structures. Combining genetics, cryosectioning and fluorescence microscopy of macrocolony biofilms, we demonstrate here in situ that this local matrix heterogeneity is generated by a c-di-GMP-dependent molecular switch characterized by several nested positive and negative feedback loops. In this switch, the trigger phosphodiesterase PdeR is the key component for establishing local heterogeneity in the activation of the transcription factor MlrA, which in turn activates expression of the major matrix regulator CsgD. Upon its release of direct inhibition by PdeR, the second switch component, the diguanylate cyclase DgcM, activates MlrA by direct interaction. Antagonistically acting PdeH and DgcE provide for a PdeR-sensed c-di-GMP input into this switch and-via their spatially differentially controlled expression-generate the long-range vertical asymmetry of the matrix architecture. Using flow cytometry, we show heterogeneity of CsgD expression to also occur in spatially unstructured planktonic cultures, where it is controlled by the same c-di-GMP circuitry as in macrocolony biofilms. Quantification by flow cytometry also showed CsgD subpopulations with distinct CsgD expression levels and revealed an additional fine-tuning feedback within the PdeR/DgcM-mediated switch that depends on c-di-GMP synthesis by DgcM. Finally, local heterogeneity of matrix production was found to be crucial for the tissue-like elasticity that allows for large-scale wrinkling and folding of macrocolony biofilms.
Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB.
Nieto Vincent,Partridge Jonathan D,Severin Geoffrey B,Lai Run-Zhi,Waters Christopher M,Parkinson John S,Harshey Rasika M
Journal of bacteriology
In and , the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of restores wild-type motor behavior in , indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In , c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in Instead, we found through reversion analysis that deletion of , which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σ for degradation, restored wild-type motility in the mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression. Flagellum-driven motility has been studied in and for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in , we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σ regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.
CRISPR-Cas9 knockout of induced asynchrony between motility and biofilm formation in .
Gou Yi,Liu Weiqi,Wang Jing Jing,Tan Ling,Hong Bin,Guo Linxia,Liu Haiquan,Pan Yingjie,Zhao Yong
Canadian journal of microbiology
Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the MG1655Δ strain (strain Δ), and the effects of the gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the Δ strain had higher ( < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the Δ and WT strains. Real-time quantitative PCR illustrated that deletion of in the WT strain downregulated expression of the type I pili gene . Therefore, we might conclude that the Δ induced the downregulation of , which led to asynchrony between motility and biofilm formation in , providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.
Biogenic synthesis of silver nanoparticles using Piper betle aqueous extract and evaluation of its anti-quorum sensing and antibiofilm potential against uropathogens with cytotoxic effects: an in vitro and in vivo approach.
Srinivasan Ramanathan,Vigneshwari Loganathan,Rajavel Tamilselvam,Durgadevi Ravindran,Kannappan Arunachalam,Balamurugan Krishnaswamy,Pandima Devi Kasi,Veera Ravi Arumugam
Environmental science and pollution research international
Urinary tract infections are the utmost common bacterial infections caused by Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, and Serratia marcescens. These uropathogens resist the action of several antibiotics due to their ability to form biofilms. Most of these bacterial pathogens use the quorum sensing (QS) machinery to co-ordinate their cells and regulate several virulence factors and biofilm formation. On the other hand, the anti-quorum sensing (anti-QS) and antibiofilm potential of silver nanoparticles have been well reported against certain bacterial pathogens, but to the best of our knowledge, no report is available against the pathogenicity of uropathogens in particular S. marcescens and P. mirabilis. Therefore, the present study is primarily focused on the anti-QS and antibiofilm potential of Piper betle-based synthesized silver nanoparticles (PbAgNPs) against S. marcescens and P. mirabilis. Initially, the silver nanoparticles were synthesized by the aqueous extract of P. betle and characterized by UV-absorbance spectroscopy, XRD, FT-IR, SEM, TEM, and DLS. The synthesized silver nanoparticles were assessed for their anti-QS activity and the obtained results revealed that the PbAgNPs inhibited the QS-mediated virulence factors such as prodigiosin, protease, biofilm formation, exopolysaccharides and hydrophobicity productions in uropathogens. The gene expression analysis divulged the downregulation of fimA, fimC, flhD, and bsmB genes in S. marcescens and flhB, flhD, and rsbA genes in P. mirabilis, respectively. The in vivo Caenorhabditis elegans assays revealed the non-toxic and anti-adherence efficiency of PbAgNPs. Furthermore, the non-toxic effect of PbAgNPs was also confirmed through peripheral blood mononuclear cells and normal lung epithelial cells. Therefore, the contemporary study demonstrates the use of PbAgNPs as a possible alternative toward conventional antibiotics in controlling QS and biofilm-related uropathogen infections.
Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli.
DiBiasio Eric C,Ranson Hilary J,Johnson James R,Rowley David C,Cohen Paul S,Camberg Jodi L
Journal of bacteriology
Uropathogenic (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. Uropathogenic (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.
The specific effect of gallic acid on Escherichia coli biofilm formation by regulating pgaABCD genes expression.
Kang Jiamu,Li Qianqian,Liu Liu,Jin Wenyuan,Wang Jingfan,Sun Yuyang
Applied microbiology and biotechnology
Escherichia coli (E. coli) is associated with an array of health-threatening contaminations, some of which are related to biofilm states. The pgaABCD-encoded poly-beta-1,6-N-acetyl-D-glucosamine (PGA) polymer plays an important role in biofilm formation. This study was conducted to determine the inhibitory effect of gallic acid (GA) against E. coli biofilm formation. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of GA against planktonic E. coli were 0.5 and 4 mg/mL, and minimal biofilm inhibitory concentration and minimal biofilm eradication concentration values of GA against E. coli in biofilms were 2 and 8 mg/mL, respectively. Quantitative crystal violet staining of biofilms and ESEM images clearly indicate that GA effectively, dose-dependently inhibited biofilm formation. CFU counting and confocal laser scanning microscopy measurements showed that GA significantly reduced viable bacteria in the biofilm. The contents of polysaccharide slime, protein, and DNA in the E. coli biofilm also decreased. qRT-PCR data showed that at the sub-MIC level of GA (0.25 mg/mL) and expression of pgaABC genes was downregulated, while pgaD gene expression was upregulated. The sub-MBC level of GA (2 mg/mL) significantly suppressed the pgaABCD genes. Our results altogether demonstrate that GA inhibited viable bacteria and E. coli biofilm formation, marking a novel approach to the prevention and treatment of biofilm-related infections in the food industry.
Repurposing auranofin to combat uropathogenic Escherichia coli biofilms.
Jang H-I,Eom Y-B
Journal of applied microbiology
AIMS:The aim of this study was to assess anti-biofilm and antimicrobial effects of auranofin, an anti-rheumatic agent, on uropathogenic Escherichia coli (UPEC) biofilm formation. METHODS AND RESULTS:The minimum inhibitory concentration and biofilm inhibition concentration of auranofin against UPEC ranged from 24 to 32 μg ml . Biofilm eradication concentration and XTT reduction concentration of auranofin were found to be at 112 μg ml . Confocal laser scanning microscopy results confirmed that biofilm was inhibited by auranofin. These results indicate that auranofin possesses potent anti-biofilm and antimicrobial activities against UPEC. Effects of auranofin on type 1 fimbriae gene (fimH) and response regulator gene (rpoS) to stress were explored using quantitative real time-polymerase chain reaction. In addition, combination of auranofin and tetracycline showed synergistic effect. CONCLUSIONS:These data indicate that auranofin has inhibitory effect on biofilm formation and synergistic effect on UPEC infection when it is combined with tetracycline. SIGNIFICANCE AND IMPACT OF THE STUDY:Our study strongly suggest that auranofin is a promising alternative anti-biofilm and antimicrobial agent to prevent UPEC biofilm formation in UTIs. Auranofin already approved for human use have the advantage of being able to be put into clinical use relatively quickly.
Microbiology of wound infection after oral cancer surgery.
Belusic-Gobic Margita,Zubovic Arijan,Predrijevac Anamarija,Harmicar David,Cerovic Robert,Udovic Gobic Silvana,Zubovic Lorena
Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery
INTRODUCTION:The aim of present study is to investigate the most common infection pathogen found in the postoperative wounds, following surgical treatment of oral and oropharyngeal cancer, in order to identify the most suitable antibiotic treatment. PATIENTS AND METHODS:We analyzed patients with squamous cell cancer of oral and oropharyngeal region. In patients who developed postoperative wound infection, wound swabs were taken from three different sites: the cannula, wounds on the neck and wounds in the oral cavity. RESULTS:In total 195 patients were included. The postoperative wound infection was detected in 115 patients (59%). In average, the swabs were taken 8 days after the surgery. The similar bacterial species from all three sites were detected in 24 patients (12,3%). In comparison, we found that there was statistically significant difference in the bacteria abundance from all three sites (p=0,031). There were significantly more bacteria in the wounds of the neck than cannula (p=0,007) and in the wounds in the oral cavity than cannula (p=0,002). No statistically significant difference between the wound on the neck and in the oral cavity was found. The most frequently isolated bacterial family was Enterobacteriaceae. Other more commonly isolated bacteria species were Staphylococcus spp. (G+), Pseudomonas aeruginosa (G-), Corynebacteruim spp. (G +) and Acinetobacter baumanii (G-). CONCLUSION:Based on the most commonly isolated groups of pathogens we concluded that probably the best empiric antibiotic treatment of wound infections until antibiogram is completed might be achieved from the group of aminoglycosides or quinolones. Antibiotic therapy should be reviewed if necessary when antibiogram is completed.
Etiology of bacterial vaginosis and polymicrobial biofilm formation.
Jung Hyun-Sul,Ehlers Marthie M,Lombaard Hennie,Redelinghuys Mathys J,Kock Marleen M
Critical reviews in microbiology
Microorganisms in nature rarely exist in a planktonic form, but in the form of biofilms. Biofilms have been identified as the cause of many chronic and persistent infections and have been implicated in the etiology of bacterial vaginosis (BV). Bacterial vaginosis is the most common form of vaginal infection in women of reproductive age. Similar to other biofilm infections, BV biofilms protect the BV-related bacteria against antibiotics and cause recurrent BV. In this review, an overview of BV-related bacteria, conceptual models and the stages involved in the polymicrobial BV biofilm formation will be discussed.
Topical povidone iodine inhibits bacterial growth in the oral cavity of patients on mechanical ventilation: a randomized controlled study.
Tsuda Shoma,Soutome Sakiko,Hayashida Saki,Funahara Madoka,Yanamoto Souichi,Umeda Masahiro
BMC oral health
BACKGROUND:Topical 0.12% chlorhexidine has been used widely to prevent ventilator-associated pneumonia in patients undergoing mechanical ventilation. However, it is not approved for mucosal application in Japan. The aims of this study were to investigate if topical povidone iodine (i) inhibits bacterial growth and (ii) disrupts the balance of the oral microbiota. METHODS:This randomized controlled clinical trial included 23 patients who underwent mechanical ventilation in the intensive care unit. The patients were divided randomly into two groups: the intervention group (n = 16) and the control group (n = 7). All patients received oral cleaning with 3% hydrogen peroxide, followed by irrigation with tap water. The patients in the intervention group received 10% povidone iodine applied topically to the oral cavity. The concentration of total bacteria in the oropharyngeal fluid were determined before, immediately after, 1 h, 2 h, and 3 h after oral care using the Rapid Oral Bacteria Quantification System, which is based on dielectrophoresis and impedance measurements. The number of streptococci, methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Porphyromonas gingivalis, and Candida albicans before, immediately after, 1 h, and 3 h after oral care were estimated based on real-time polymerase chain reaction data. RESULTS:After irrigation of the oral cavity, the number of bacteria decreased, but increased again at 1 h after oral care in the control group; however, in the intervention group, the concentration of bacteria was significantly lower than that in the control group at 1 hour (p = 0.009), 2 h (p = 0.001), and 3 h (p = 0.001) after oral care. The growth of all bacterial species tested was inhibited in the intervention group at 3 h after oral care, suggesting that povidone iodine did not disturb the balance of the oral microbiota. CONCLUSIONS:Topical application of povidone iodine after cleaning and irrigation of the oral cavity inhibited bacterial growth in the oropharyngeal fluid of patients on mechanical ventilation while not disrupting the balance of the oral microbiota. TRIAL REGISTRATION:University Hospitals Medical Information Network Clinical Trials Registry (UMIN-CTR), UMIN000028307. Registered 1 September 2017.
Superhydrophobic, nanotextured polyvinyl chloride films for delaying Pseudomonas aeruginosa attachment to intubation tubes and medical plastics.
Loo Ching-Yee,Young Paul M,Lee Wing-Hin,Cavaliere Rosalia,Whitchurch Cynthia B,Rohanizadeh Ramin
Bacterial attachment onto the surface of polymers in medical devices such as polyvinyl chloride (PVC) is influenced by the physicochemical properties of the polymer, including its surface hydrophobicity and roughness. In this study, to prevent biofilm formation onto PVC devices, the PVC surface was modified using a combination of solvent (tetrahydrofuran) and non-solvents (i.e. ethanol and methanol). The surface of unmodified PVC was smooth and relatively hydrophobic (water contact angle (CA)=80°). Ethanol-treated PVCs revealed the presence of micron-sized particulates and porous structures as the concentration of ethanol was increased. Surface hydrophobicity (measured in terms of CA) increased from 73° to 150° as the ethanol concentration increased from 15% to 35% (v/v). In general, methanol-treated PVCs were more hydrophilic compared to those treated with ethanol. The colonization of Pseudomonas aeruginosa PAO1 onto unmodified PVC surface was rapid, and individual bacterial cells could be seen after 6h incubation. On the surface of treated PVC, the secretion of extracellular matrix layers was evident at 18 h and P. aeruginosa PAO1 start to form microcolonies at 24h of incubation. The initial attachment of P. aeruginosa PAO1 was delayed to 18 and 24h, respectively in the PVCs treated with 25% (v/v) and 35% (v/v) ethanol. It can be concluded that the treatment used in this study to prepare superhydrophobic PVC surface prevented the colonization of bacteria up to 24h after culture.
Autoinducer production and quorum-sensing dependent phenotypes of Pseudomonas aeruginosa vary according to isolation site during colonization of intubated patients.
Favre-Bonté Sabine,Chamot Eric,Köhler Thilo,Romand Jacques-A,van Delden Christian
BACKGROUND:Pseudomonas aeruginosa frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules (autoinducers, AIs) 3-oxo-C12-HSL and C4-HSL, regulates the production by P. aeruginosa of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear. RESULTS:We wondered whether QS is active during colonization of intubated patients, and whether P. aeruginosa isolates growing inside the biofilm covering the intubation devices and those resident in the lungs of colonized patients differ in their QS-dependent phenotypes. We collected the intubation devices of eight patients colonized by P. aeruginosa. We detected 3-oxo-C12-HSL on eight, and C4-HSL on six of these devices. In three of these patients we also obtained P. aeruginosa isolates from tracheal aspirates at the time of extubation (n = 18), as well as isolates from the intubation devices (n = 25). We genotyped these isolates, quantified their AIs production, and determined three QS-dependent phenotypes (adherence capacity, biofilm and elastase production). The production of 3-oxo-C12-HSL was consistently increased for isolates from the intubation devices, whereas the production of C4-HSL was significantly higher for isolates from tracheal aspirates. Isolates from tracheal aspirates produced significantly higher amounts of elastase but less biofilm, and had a marginally reduced adhesion capacity than isolates from the intubation devices. Levels of 3-oxo-C12-HSL and elastase production correlated statistically for tracheal intubation isolates, whereas levels of 3-oxo-C12-HSL production and adhesion ability, as well as biofilm production, correlated weakly amongst intubation device isolates. CONCLUSION:Our findings demonstrate that autoinducers are produced during the colonization of intubated patients by P. aeruginosa. The microenvironment, in which P. aeruginosa grows, may select for bacteria with different capacities to produce autoinducers and certain QS-dependent phenotypes. QS-inhibition might therefore affect differently isolates growing inside the biofilm covering intubation devices and those resident in the lungs.
Antimicrobial-coated endotracheal tubes: an experimental study.
Berra Lorenzo,Curto Francesco,Li Bassi Gianluigi,Laquerriere Patrice,Pitts Betsey,Baccarelli Andrea,Kolobow Theodor
Intensive care medicine
OBJECTIVE:Antibiotic-resistant bacterial biofilm may quickly form on endotracheal tubes (ETTs) and can enter the lungs, potentially causing pneumonia. In an attempt to prevent bacterial colonization, we developed and tested in an in-vitro study and animal study several antibacterial-coated ETTs (silver sulfadiazine with and without carbon in polyurethane, silver sulfadiazine and chlorhexidine with and without carbon in polyurethane, silver-platinum with and without carbon in polyurethane, chlorhexidine in polyurethane, and rose bengal for UV light). DESIGN, SETTING, ANIMALS, INTERVENTIONS: After preliminary studies, silver sulfadiazine in polyurethane (SSD-ETT) was selected among the coatings to be challenged every 24 h with 10(4)-10(6) Pseudomonas aeruginosa/ml and evaluated at 6 h, 24 h, and 72 h with standard microbiological studies, scanning electron microscopy, and confocal scanning microscopy. Subsequently, eight sheep were randomized to receive either a SSD-ETT or a standard ETT (St-ETT). After 24 h of mechanical ventilation, standard microbiological studies were performed together with scanning electron microscopy and confocal microscopy. MEASUREMENTS AND RESULTS:In the in-vitro study SSD-ETT remained bacteria-free for up to 72 h, whereas St-ETT showed heavy P. aeruginosa growth and biofilm formation (p < 0.01). In sheep, the SSD-ETT group showed no bacterial growth in the ETT, ventilator tubing, and lower respiratory tract, while heavy colonization was found in the St-ETT (p < 0.01), ventilator tubing (p=0.03), and lower respiratory tract (p < 0.01). CONCLUSION:This study describes several effective and durable antibacterial coatings for ETTs. Particularly, SSD-ETT showed prevention against P. aeruginosa biofilm formation in a 72-h in-vitro study and lower respiratory tract colonization in sheep mechanically ventilated for 24 h.
Bactericidal effects of silver plus titanium dioxide-coated endotracheal tubes on Pseudomonas aeruginosa and Staphylococcus aureus.
Tarquinio Keiko M,Kothurkar Nikhil K,Goswami Dharendra Y,Sanders Ronald C,Zaritsky Arno L,LeVine Ann Marie
International journal of nanomedicine
PURPOSE:Ventilator-associated pneumonia (VAP) is a nosocomial infection resulting in significant morbidity and mortality. Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are pathogens associated with VAP. Silver (Ag) coating of endotracheal tubes (ETTs) reduces bacterial colonization, however titanium dioxide (TiO(2)) coating has not been studied. METHODS:Five types of ETT coatings were applied over silica layer: Ag, solgel TiO(2), solgel TiO(2) with Ag, Degussa P25 TiO(2) (Degussa TiO(2)), and Degussa TiO(2) with Ag. After ETTs were incubated with P. aeruginosa or S. aureus; colonization was determined quantitatively. RESULTS:Pseudomonas aeruginosa and S. aureus grew for 5 days on standard ETTs. Compared to standard ETTs, P. aeruginosa growth was significantly inhibited by solgel TiO(2) with Ag at 24 hours, and by Degussa TiO(2) with Ag at 24 and 48 hours after inoculation. No significant difference in S. aureus growth was observed between the control and any of the five coatings for 5 days. CONCLUSION:In vitro, solgel TiO(2) with Ag and Degussa TiO(2) with Ag both attenuated P. aeruginosa growth, but demonstrated no effect on S. aureus colonization. Further studies using alternative coating and incorporating UV light exposure are needed to identify their potential utility in reducing VAP.
Ventilator-associated pneumonia: The central role of transcolonization.
Soussan Romy,Schimpf Caroline,Pilmis Benoît,Degroote Thècle,Tran Marc,Bruel Cédric,Philippart François,
Journal of critical care
Ventilator-associated pneumonia remain frequent and serious diseases since they are associated with considerable crude mortality. Pathophysiology is centered on modifications of regional bacterial flora, especially tracheobronchial tree and oropharyngeal sphere. Bacterial migration from an anatomical area to another seems to be the main explanation of these alterations which are called "transcolonization". The association of transcolonization and lack of tightness of the endotracheal tube cuff provides a direct pathway for bacteria from the upper to the subglottic airways, eventually leading to ventilator-associated pneumonia. Although modification of bacterial flora has been largely studied, the mechanism which underlays the ability of the implantation, growing and interactions with the local microbiome that leads to the observed transcolonization remains to be more clearly deciphered. The aim of our review is to emphasize the cornerstone importance of the "transcolonization" as a nosological entity playing a central role in ventilator-associated pneumonia.
Endotracheal tube biofilm and ventilator-associated pneumonia with mechanical ventilation.
De Souza Paula Regina,De Andrade Denise,Cabral Danielle Bezerra,Watanabe Evandro
Microscopy research and technique
OBJECTIVE:To analyze biofilm on internal and external surfaces of endotracheal tubes after their use in critical care patients, and to produce evidence of association between use of the tube, presence of biofilm, and the occurrence of pneumonia. METHODS:This was a clinical study performed at the Intensive Care Unit of an emergency hospital in the interior of São Paulo state, Brazil. Data collection involved 30 endotracheal tubes used on adult patients for a period of ≥48 h of mechanical ventilation for scanning electron microscopy. RESULTS:Analysis of the biofilm on the 30 tubes by scanning electron microscopy showed various abiotic and biotic structures, predominantly on the internal surface, such as: fibrin network, erythrocytes, leukocytes, cocci, bacilli, and molds, among others. The intubation period of the endotracheal tube for ≥8 days represented one of the risk factors for ventilator-associated pneumonia (RR 7.41, P < 0.001). CONCLUSIONS:The presence of the endotracheal tube permits microbial colonization, overall contributing to the development of biofilm and the occurrence of pneumonia.
Internally coated endotracheal tubes with silver sulfadiazine in polyurethane to prevent bacterial colonization: a clinical trial.
Berra Lorenzo,Kolobow Theodor,Laquerriere Patrice,Pitts Betsey,Bramati Simone,Pohlmann Joshua,Marelli Chiara,Panzeri Miriam,Brambillasca Pietro,Villa Federico,Baccarelli Andrea,Bouthors Sylvie,Stelfox Henry T,Bigatello Luca M,Moss Joel,Pesenti Antonio
Intensive care medicine
OBJECTIVE:Coated medical devices have been shown to reduce catheter-related infections. We coated endotracheal tubes (ETT) with silver sulfadiazine (SSD), and tested them in a clinical study to assess the feasibility, safety, and efficacy of preventing bacterial colonization. DESIGN:A prospective, randomized clinical trial, phase I-II. SETTING:Academic intensive care unit (ICU). PARTICIPANTS:Forty-six adult patients expected to need 12-24 h of intubation were randomized into two groups. INTERVENTIONS:Patients were randomized to be intubated with a standard non-coated ETT (St-ETT, n=23; control group), or with a SSD-coated ETT (SSD-ETT, n=23). MEASUREMENTS AND RESULTS:Coating with SSD prevented bacterial colonization of the ETT (frequency of colonization: SSD-ETT 0/23, St-ETT 8/23; p<0.01). No organized bacterial biofilm could be identified on the lumen of any ETT; however, SSD was associated with a thinner mucus layer (in the SSD-ETT secretion deposits ranged from 0 to 200 microm; in the St-ETT deposits ranged between 50 and 700 microm). No difference was observed between the two groups in the tracheobronchial brush samples (frequency of colonization: SSD-ETT 0/23, St-ETT 2/23; p=0.48). No adverse reactions were observed with the implementation of the novel device. CONCLUSION:SSD-ETT can be safely used in preventing bacterial colonization and narrowing of the ETT in patients intubated for up to 24 h (mean intubation time 16 h).
Green fabrication of anti-bacterial biofilm layer on endotracheal tubing using silver nanoparticles embedded in polyelectrolyte multilayered film.
Daengngam Chalongrat,Lethongkam Sakkarin,Srisamran Panuwat,Paosen Supakit,Wintachai Phitchayapak,Anantravanit Bodin,Vattanavanit Veerapong,Voravuthikunchai Supayang
Materials science & engineering. C, Materials for biological applications
Endotracheal tubes (ETTs) are a common source of bacterial colonization, leading to ventilator-associated pneumonia (VAP). This research developed a biofilm-resistant ETT, following the principles of green chemistry. Using an aqueous layer-by-layer (LbL) technique, a thick polyelectrolyte multilayered film was deposited on a ventilation tube. The polyelectrolyte multilayered film accommodated silver nanoparticles (AgNPs) formed in situ by reducing Ag ions with Eucalyptus citriodora leaf extract. The multilayered film coating conformed to the curved surfaces of the ETT. Film thickness and silver content increased exponentially with the number of polyelectrolyte bilayer pairs, and a sufficiently high AgNPs content of 10-30%w/w was obtained at 75 to 125 bilayer films. Adhesion of the Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa was prevented by 99.9 and 99.99%, respectively, without cytotoxic effects against human lung epithelial cells (p < 0.05).
Respiratory pathogen colonization of dental plaque, the lower airways, and endotracheal tube biofilms during mechanical ventilation.
Sands Kirsty M,Wilson Melanie J,Lewis Michael A O,Wise Matt P,Palmer Nicki,Hayes Anthony J,Barnes Rosemary A,Williams David W
Journal of critical care
PURPOSE:In mechanically ventilated patients, the endotracheal tube is an essential interface between the patient and ventilator, but inadvertently, it also facilitates the development of ventilator-associated pneumonia (VAP) by subverting pulmonary host defenses. A number of investigations suggest that bacteria colonizing the oral cavity may be important in the etiology of VAP. The present study evaluated microbial changes that occurred in dental plaque and lower airways of 107 critically ill mechanically ventilated patients. MATERIALS AND METHODS:Dental plaque and lower airways fluid was collected during the course of mechanical ventilation, with additional samples of dental plaque obtained during the entirety of patients' hospital stay. RESULTS:A "microbial shift" occurred in dental plaque, with colonization by potential VAP pathogens, namely, Staphylococcus aureus and Pseudomonas aeruginosa in 35 patients. Post-extubation analyses revealed that 70% and 55% of patients whose dental plaque included S aureus and P aeruginosa, respectively, reverted back to having a predominantly normal oral microbiota. Respiratory pathogens were also isolated from the lower airways and within the endotracheal tube biofilms. CONCLUSIONS:To the best of our knowledge, this is the largest study to date exploring oral microbial changes during both mechanical ventilation and after recovery from critical illness. Based on these findings, it was apparent that during mechanical ventilation, dental plaque represents a source of potential VAP pathogens.
Inhibitory effect of a novel chicken-derived anti-biofilm peptide on P. aeruginosa biofilms and virulence factors.
Xu Dengfeng,Zhang Yang,Cheng Peng,Wang Yidong,Li Xiaofen,Wang Zhiying,Yi Huashan,Chen Hongwei
The antibiotic resistance of Pseudomonas aeruginosa (P. aeruginosa) is correlated with the formation of biofilms. Several studies have focused on biofilms and the treatment of biofilm infection by antimicrobial peptides (AMPs). The present study analyzed the feasibility of cCATH-2 (a chicken-derived antimicrobial peptide) as a new strategy for anti-biofilm activities. Biofilm biomass (crystal violet staining) and viability of biofilm bacteria (colony counting) were measured in P. aeruginosa PAO1 biofilm at the stage of attachment (4 h), formation (14 h), and maturation (24 h). cCATH-2 (1/2MIC) had the ability to reduce the initial attachment of viable bacteria due to decreasing planktonic bacteria. All tested concentrations of cCATH-2 (1/32-1/2MIC) significantly reduced the biomass at the biofilm formation stage. In addition, cCATH-2 (2MIC) had significant effects on the biomass and viability of bacteria of pre-biofilms, which caused significant killing (>90%) of the bacteria in the biofilm. Thus, it was confirmed that cCATH-2 could infiltrate into pre-biofilm to kill the biofilm cells, as assessed by confocal laser scanning microscopy (CLSM). Furthermore, cCATH-2 had an obvious effect on the production of the majority of the virulence factors of PAO1 biofilms, and the effect was better than that of ciprofloxacin, especially on alginate (the structural component of biofilms). These findings suggested that cCATH-2 is a putative candidate for the development of anti-biofilm and anti-infective drugs.
Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm.
Martins Carlos Henrique Gomes,Pires Regina Helena,Cunha Aline Oliveira,Pereira Cristiane Aparecida Martins,Singulani Junya de Lacorte,Abrão Fariza,Moraes Thais de,Mendes-Giannini Maria José Soares
Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher (p < 0.001) than non-albicans Candida strains, after 6 h 37 °C. The total C. albicans CFU from a dual-species biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans/non-albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm.
Aloe-emodin inhibits Staphylococcus aureus biofilms and extracellular protein production at the initial adhesion stage of biofilm development.
Xiang Hua,Cao Fengjiao,Ming Di,Zheng Yanyang,Dong Xiaoyun,Zhong Xiaobo,Mu Dan,Li Bangbang,Zhong Ling,Cao Junjie,Wang Lin,Ma Hongxia,Wang Tiedong,Wang Dacheng
Applied microbiology and biotechnology
Staphylococcus aureus (S. aureus) biofilms are clinically serious and play a critical role in the persistence of chronic infections due to their ability to resist antibiotics. The inhibition of biofilm formation is viewed as a new strategy for the prevention of S. aureus infections. Here, we demonstrated that minimum inhibitory concentrations (MICs) of aloe-emodin exhibited no bactericidal activity against S. aureus but affected S. aureus biofilm development in a dose-dependent manner. Further studies indicated that aloe-emodin specifically inhibits the initial adhesion and proliferation stages of S. aureus biofilm development. Scanning electron microscopy (SEM) indicated that the S. aureus ATCC29213 biofilm extracellular matrix is mainly composed of protein. Laser scanning confocal microscope assays revealed that aloe-emodin treatment primarily inhibited extracellular protein production. Moreover, the Congo red assay showed that aloe-emodin also reduced the accumulation of polysaccharide intercellular adhesin (PIA) on the cell surface. These findings will provide new insights into the mode of action of aloe-emodin in the treatment of infections by S. aureus biofilms.
The role of biofilms in persistent infections and factors involved in ica-independent biofilm development and gene regulation in Staphylococcus aureus.
Figueiredo Agnes Marie Sá,Ferreira Fabienne Antunes,Beltrame Cristiana Ossaille,Côrtes Marina Farrel
Critical reviews in microbiology
Staphylococcus aureus biofilms represent a unique micro-environment that directly contribute to the bacterial fitness within hospital settings. The accumulation of this structure on implanted medical devices has frequently caused the development of persistent and chronic S. aureus-associated infections, which represent an important social and economic burden worldwide. ica-independent biofilms are composed of an assortment of bacterial products and modulated by a multifaceted and overlapping regulatory network; therefore, biofilm composition can vary among S. aureus strains. In the microniches formed by biofilms-produced by a number of bacterial species and composed by different structural components-drug refractory cell subpopulations with distinct physiological characteristics can emerge and result in therapeutic failures in patients with recalcitrant bacterial infections. In this review, we highlight the importance of biofilms in the development of persistence and chronicity in some S. aureus diseases, the main molecules associated with ica-independent biofilm development and the regulatory mechanisms that modulate ica-independent biofilm production, accumulation, and dispersion.
Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action.
Roy Ranita,Tiwari Monalisa,Donelli Gianfranco,Tiwari Vishvanath
Biofilm refers to the complex, sessile communities of microbes found either attached to a surface or buried firmly in an extracellular matrix as aggregates. The biofilm matrix surrounding bacteria makes them tolerant to harsh conditions and resistant to antibacterial treatments. Moreover, the biofilms are responsible for causing a broad range of chronic diseases and due to the emergence of antibiotic resistance in bacteria it has really become difficult to treat them with efficacy. Furthermore, the antibiotics available till date are ineffective for treating these biofilm related infections due to their higher values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), which may result in in-vivo toxicity. Hence, it is critically important to design or screen anti-biofilm molecules that can effectively minimize and eradicate biofilm related infections. In the present article, we have highlighted the mechanism of biofilm formation with reference to different models and various methods used for biofilm detection. A major focus has been put on various anti-biofilm molecules discovered or tested till date which may include herbal active compounds, chelating agents, peptide antibiotics, lantibiotics and synthetic chemical compounds along with their structures, mechanism of action and their respective MICs, MBCs, minimum biofilm inhibitory concentrations (MBICs) as well as the half maximal inhibitory concentration (IC) values available in the literature so far. Different mode of action of anti biofilm molecules addressed here are inhibition via interference in the quorum sensing pathways, adhesion mechanism, disruption of extracellular DNA, protein, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways. From this study, we conclude that the molecules considered here might be used to treat biofilm-associated infections after significant structural modifications, thereby investigating its effective delivery in the host. It should also be ensured that minimum effective concentration of these molecules must be capable of eradicating biofilm infections with maximum potency without posing any adverse side effects on the host.
Evaluation of combinations of putative anti-biofilm agents and antibiotics to eradicate biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.
Belfield Katherine,Bayston Roger,Hajduk Nadzieja,Levell Georgia,Birchall John P,Daniel Matija
The Journal of antimicrobial chemotherapy
Objectives:To evaluate potential anti-biofilm agents for their ability to enhance the activity of antibiotics for local treatment of localized biofilm infections. Methods:Staphylococcus aureus and Pseudomonas aeruginosa in vitro biofilm models were developed. The putative antibiotic enhancers N-acetylcysteine, acetylsalicylic acid, sodium salicylate, recombinant human deoxyribonuclease I, dispersin B, hydrogen peroxide and Johnson's Baby Shampoo (JBS) were tested for their anti-biofilm activity alone and their ability to enhance the activity of antibiotics for 7 or 14 days, against 5 day old biofilms. The antibiotic enhancers were paired with rifampicin and clindamycin against S. aureus and gentamicin and ciprofloxacin against P. aeruginosa. Isolates from biofilms that were not eradicated were tested for antibiotic resistance. Results:Antibiotic levels 10× MIC and 100× MIC significantly reduced biofilm, but did not consistently eradicate it. Antibiotics at 100× MIC with 10% JBS for 14 days was the only treatment to eradicate both staphylococcal and pseudomonal biofilms. Recombinant human deoxyribonuclease I significantly reduced staphylococcal biofilm. Emergence of resistance of surviving isolates was minimal and was often associated with the small colony variant phenotype. Conclusions:JBS enhanced the activity of antibiotics and several other promising anti-biofilm agents were identified. Antibiotics with 10% JBS eradicated biofilms produced by both organisms. Such combinations might be useful in local treatment of localized biofilm infections.
Suspected periprosthetic joint infection after total knee arthroplasty under propofol versus sevoflurane anesthesia: a retrospective cohort study.
Kishimoto Miwa,Yamana Hayato,Inoue Satoki,Noda Tatsuya,Akahane Manabu,Inagaki Yusuke,Matsui Hiroki,Yasunaga Hideo,Kawaguchi Masahiko,Imamura Tomoaki
Canadian journal of anaesthesia = Journal canadien d'anesthesie
PURPOSE:Periprosthetic joint infection is a serious complication of total knee arthroplasty. Though there are many factors that might increase its risk, the use of propofol for maintaining general anesthesia could theoretically increase the incidence of infection because of its lipid component that supports bacterial growth. Nevertheless, the relationship between anesthetic maintenance agents and the occurrence of periprosthetic joint infection remains uncertain. The purpose of this study was to compare the incidence of suspected early-onset periprosthetic joint infection between patients undergoing total knee arthroplasty under propofol vs sevoflurane anesthesia. METHODS:We conducted a retrospective cohort study of patients in the national inpatient Diagnosis Procedure Combination database in Japan who underwent total knee arthroplasty. Suspected periprosthetic joint infection was surrogately defined as the need for arthrocentesis or debridement within 30 days of surgery. Propensity score matching was performed between patients who received either propofol or sevoflurane for anesthetic maintenance to determine the proportion of those with infection. RESULTS:Eligible patients (n = 21,899) were categorized into either the propofol (n = 7,439) or sevoflurane (n = 14,460) groups. In the 5,140 propensity-matched patient pairs, there was no significant difference in the proportion of arthrocentesis or debridement [1.3% propofol vs 1.7% sevoflurane; respectively (relative risk, 0.76; 95% CI, 0.55 to 1.04; P = 0.10)] between the groups. The mean (SD) length of stay in the propofol group was significantly longer than in the sevoflurane group [32.5 (18.4) days vs 31.4 (14.4) days, respectively; mean difference, 1.1; 95% CI, 0.5 to 1.8; P < 0.001]. CONCLUSION:Propensity score analysis suggested no significant association between the choice of anesthetic maintenance agent and the occurrence of suspected early-onset periprosthetic joint infection in patients undergoing total knee arthroplasty.
Commensal Microbiota Promote Lung Cancer Development via γδ T Cells.
Jin Chengcheng,Lagoudas Georgia K,Zhao Chen,Bullman Susan,Bhutkar Arjun,Hu Bo,Ameh Samuel,Sandel Demi,Liang Xu Sue,Mazzilli Sarah,Whary Mark T,Meyerson Matthew,Germain Ronald,Blainey Paul C,Fox James G,Jacks Tyler
Lung cancer is closely associated with chronic inflammation, but the causes of inflammation and the specific immune mediators have not been fully elucidated. The lung is a mucosal tissue colonized by a diverse bacterial community, and pulmonary infections commonly present in lung cancer patients are linked to clinical outcomes. Here, we provide evidence that local microbiota provoke inflammation associated with lung adenocarcinoma by activating lung-resident γδ T cells. Germ-free or antibiotic-treated mice were significantly protected from lung cancer development induced by Kras mutation and p53 loss. Mechanistically, commensal bacteria stimulated Myd88-dependent IL-1β and IL-23 production from myeloid cells, inducing proliferation and activation of Vγ6Vδ1 γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. Our findings clearly link local microbiota-immune crosstalk to lung tumor development and thereby define key cellular and molecular mediators that may serve as effective targets in lung cancer intervention.
Gram-negative bacteria facilitate tumor progression through TLR4/IL-33 pathway in patients with non-small-cell lung cancer.
Sun Mengyao,Bai Yang,Zhao Song,Liu Xiyu,Gao Yongsheng,Wang Lei,Liu Bin,Ma Dashi,Ma Chunye
Non-small-cell lung cancer (NSCLC) accounts for the most cases in clinical lung cancer patients. Patients with NSCLC are often diagnosed in advanced stage and frequently infected with gram-negative bacteria. Pulmonary infection with gram-negative bacteria is the most frequent postoperative complication in NSCLC patients. While accumulating evidence indicate an involvement of gram-negative bacteria in NSCLC progression, the underlying mechanisms remain largely unknown. Herein, we explored the effect of gram-negative bacteria on tumor progression using tumor cells from NSCLC patients. We observed that infection with gram-negative bacteria predicted advanced stages and decreased time interval to recurrence of NSCLC patients. Incubation of NSCLC cells with gram-negative bacteria promoted their growth and metastasis. Mechanistically, gram-negative bacteria activated Toll-like receptor 4 (TLR4) signaling in NSCLC cells, leading to increased mRNA and protein expression of interleukin 33 (IL-33) through MyD88-dependent pathway. Knockdown of IL-33 abrogated the contribution of gram-negative bacteria to NSCLC progression by regulating cancer metabolic activities and stem cell properties. In NSCLC patients, higher TLR4 expression was associated with increased IL-33 expression, Ki-67 proliferation index and CD133 expression in those with gram-negative bacterial infection. These findings shed new light on the molecular mechanisms underlie gram-negative bacteria mediated tumor progression and provide clues for innovative therapeutic explorations for NSCLC patients.
Mycobacterium tuberculosis H37Rv infected THP-1 cells induce epithelial mesenchymal transition (EMT) in lung adenocarcinoma epithelial cell line (A549).
Gupta Pramod Kumar,Tripathi Devavrat,Kulkarni Savita,Rajan M G R
Chronic infections of Mycobacterium tuberculosis (MTB) cause oxidative stress, TLR activation and production of inflammatory cytokines and thus can create an environment reinforcing tumorigenesis, progression and metastasis. Epidemiological studies have established a relation between lung cancer and tuberculosis but cellular mechanism is still poorly understood. In present study, we have shown for the first time that MTB infection in human monocytic cell line (THP-1) enhances invasion and induces EMT characteristics in lung adenocarcinoma cell line (A549) during co-culture. After co-culture with MTB infected THP-1 cells A549 cells exhibited morphological and molecular signatures of EMT. During co-culture, expression of inflammatory cytokines like TNF-α, IL-1β and IL-6 was enhanced in the microenvironment of A549 cells in comparison to single culture of A549 cells. Using pharmacological inhibitors of JNK (SP-600125) and p38 MAPK (SB-203580), we demonstrated the involvement of JNK and p38 MAPK in MTB induced EMT induction in A549 cells. To the best of our knowledge this is the first report demonstrating the role of MTB infection in induction of metastasis associated EMT in lung cancer.
[RL-RVG inhibits proliferation and promotes apoptosis of lung cancer cells in vitro].
Yan Yulan,Liang Bing,Zhang Jin,Jia Lijuan,Liu Yang,Zhang Jie
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
OBJECTIVE:To infect lung adenocarcinoma A549 cells with recombinant LaSota strain Newcastle disease virus (NDV) vaccine expressing the rabies virus glycoprotein (rL-RVG), and explore the effect of rL-RVG on proliferation and apoptosis of lung cancer cells. METHODS:A549 cells were infected with the rL-RVG and then detected for the expressions of RVG and NDV proteins by Western blotting. The cell proliferation was examined by MTT assay and apoptosis index and cell early apoptosis were respectively detected by TUNEL and annexin V-FITC/PI staining combined with flow cytometry. The expression of pro-apoptotic protein caspase-3 was observed by Western blotting. The LaSota strain of NDV was used as control group and PBS was the blank control. RESULTS:Both RVG and NDV proteins were stably expressed in A549 cells infected with rL-RVG. MTT assay results showed that cell proliferation was significantly inhibited, and the inhibition rate of the rL-RVG group was higher than that of LaSota group. The apoptosis of A549 cells were promoted by rL-RVG infection. Flow cytometry revealed that the early apoptotic cells of the rL-RVG group increased as compared with the other two groups (P<0.05). Consistently, TUNEL assay showed the apoptotic index increased in the rL-RVG group as compared with the other two groups (P<0.05). Western blotting demonstrated that the expression of the pro-apoptotic protein caspase-3 was up-regulated. However, when we added specific broad-spectrum caspase inhibitor Z-VAD-FMK, the expression of the caspase-3 protein was obviously reduced. CONCLUSION:The rL-RVG is stably expressed in the infected A549 cells. The rL-RVG inhibits lung cancer cell growth and promote cell apoptosis, and the effect of rL-RVG is better than the wild LaSota strain.
Loss of TIMP-3 promotes tumor invasion via elevated IL-6 production and predicts poor survival and relapse in HPV-infected non-small cell lung cancer.
Wu De-Wei,Tsai Lung-Hung,Chen Po-Ming,Lee Ming-Ching,Wang Lee,Chen Chih-Yi,Cheng Ya-Wen,Lee Huei
The American journal of pathology
Human papillomavirus (HPV) 16/18 E6 oncoprotein is expressed in lung tumors and is associated with p53 inactivation. The tissue inhibitor of metalloproteinase 3 (TIMP-3) is essential for limiting inflammation; therefore, we expected that TIMP-3 loss might induce chronic inflammation, thereby promoting tumor malignancy as well as poor survival and relapse in patients with HPV-infected non-small cell lung cancer. In this study, the loss of TIMP-3 by loss of heterozygosity and/or promoter hypermethylation was more frequent in HPV16/18 E6-positive tumors than in E6-negative tumors. To explore the possible underlying mechanism, E6-negative TL4 and CL1-0 cells were transfected with an E6 cDNA plasmid. A marked decrease in TIMP-3 expression was caused by promoter hypermethylation via increased DNA (cytosine-5-)-methyltransferase 1 (DNMT1) expression. Mechanistic studies indicated that TIMP-3 loss promoted interleukin-6 (IL-6) production, which led to cell invasion and anchorage-independent growth on soft agar plates. Kaplan-Meier and Cox regression models showed that patients with low-TIMP-3/high-IL-6 tumors had shorter overall survival and relapse-free survival periods when compared with patients with high-TIMP-3/low-IL-6 tumors. In summary, loss of TIMP-3 may increase IL-6 production via the tumor necrosis factor α/nuclear factor κB axis, thereby promoting tumor malignancy and subsequent relapse and poor survival in patients with HPV-infected non-small cell lung cancer.
Gram-positive pneumonia augments non-small cell lung cancer metastasis via host toll-like receptor 2 activation.
Gowing Stephen D,Chow Simon C,Cools-Lartigue Jonathan J,Chen Crystal B,Najmeh Sara,Jiang Henry Y,Bourdeau France,Beauchamp Annie,Mancini Ugo,Angers Isabelle,Giannias Betty,Spicer Jonathan D,Rousseau Simon,Qureshi Salman T,Ferri Lorenzo E
International journal of cancer
Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.
TREM1: A positive regulator for inflammatory response via NF-κB pathway in A549 cells infected with Mycoplasma pneumoniae.
Liu Fang,Zhang XinGuang,Zhang Bin,Mao WenWei,Liu TianTian,Sun Min,Wu YaHui
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Herein, we found that serum content of the triggering receptor expressed on myeloid cells-1 (TREM1) was increased, and positively correlated with Mycoplasma pneumoniae (MP)-DNA in children with MP infection. In this study, A549 cells, known as human lung epithelial cells, were co-cultured with 10 CCU/ml of MP to established in vitro model of MP infection. We studied the roles of TREM1 in inflammatory response of A549 cell by regulating the secretions of cytokine interleukin (IL)-8 and tumor necrosis factor (TNF)-α in cell culture supernatants. Moreover, transcriptional activity of nuclear factor kappa B (NF-кB) was assessed by measuring protein levels of NF-кB in the cytoplasm and nuclear. Our data suggested that sanguinarine chloride significantly decreased TREM1 expression, and alleviated inflammatory response of MP-infected A549 cells via preventing NF-кB nuclear translocation. To study the roles of TREM1 in inflammatory regulation in MP-infected A549 cells and the underlying mechanisms, we established TREM1 overexpression transfected A549 cells. PDTC was used for inhibiting NF-кB nuclear translocation. We found that TREM1 overexpression induced server inflammatory response of A549 cells. Besides, TREM1 overexpression attenuated anti-inflammatory effects of sanguinarine chloride in MP-infected cells. More importantly, pro-inflammatory effects of TREM1 overexpression was significantly reversed with additional PDTC treatment in MP-infected cells treated with sanguinarine chloride, suggesting that TREM1 was a pro-inflammatory factor via regulating NF-кB nuclear translocation in MP-infected A549 cells.
Commensal bacteria drive endogenous transformation and tumour stem cell marker expression through a bystander effect.
Wang Xingmin,Yang Yonghong,Huycke Mark M
OBJECTIVE:Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect. DESIGN:Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)-an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis. RESULTS:Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice. CONCLUSIONS:These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect.
Bacterial infections and cancer.
van Elsland Daphne,Neefjes Jacques
Infections are estimated to contribute to 20% of all human tumours. These are mainly caused by viruses, which explains why a direct bacterial contribution to cancer formation has been largely ignored. While epidemiological data link bacterial infections to particular cancers, tumour formation is generally assumed to be solely caused by the ensuing inflammation responses. Yet, many bacteria directly manipulate their host cell in various phases of their infection cycle. Such manipulations can affect host cell integrity and can contribute to cancer formation. We here describe how bacterial surface moieties, bacterial protein toxins and bacterial effector proteins can induce host cell DNA damage, and thereby can interfere with essential host cell signalling pathways involved in cell proliferation, apoptosis, differentiation and immune signalling.
Upregulation of virulence genes promotes biofilm hyperinfectivity.
Gallego-Hernandez A L,DePas W H,Park J H,Teschler J K,Hartmann R,Jeckel H,Drescher K,Beyhan S,Newman D K,Yildiz F H
Proceedings of the National Academy of Sciences of the United States of America
remains a major global health threat, disproportionately impacting parts of the world without adequate infrastructure and sanitation resources. In aquatic environments, exists both as planktonic cells and as biofilms, which are held together by an extracellular matrix. biofilms have been shown to be hyperinfective, but the mechanism of hyperinfectivity is unclear. Here we show that biofilm-grown cells, irrespective of the surfaces on which they are formed, are able to markedly outcompete planktonic-grown cells in the infant mouse. Using an imaging technique designed to render intestinal tissue optically transparent and preserve the spatial integrity of infected intestines, we reveal and compare three-dimensional colonization patterns of planktonic-grown and biofilm-grown cells. Quantitative image analyses show that colonizes mainly the medial portion of the small intestine and that both the abundance and localization patterns of biofilm-grown cells differ from that of planktonic-grown cells. In vitro biofilm-grown cells activate expression of the virulence cascade, including the toxin coregulated pilus (TCP), and are able to acquire the cholera toxin-carrying CTXФ phage. Overall, virulence factor gene expression is also higher in vivo when infected with biofilm-grown cells, and modulation of their regulation is sufficient to cause the biofilm hyperinfectivity phenotype. Together, these results indicate that the altered biogeography of biofilm-grown cells and their enhanced production of virulence factors in the intestine underpin the biofilm hyperinfectivity phenotype.
Circ_0026344 restrains metastasis of human colorectal cancer cells via miR-183.
Shen Tao,Cheng Xianshuo,Liu Xin,Xia Cuifeng,Zhang Hongtao,Pan Dingguo,Zhang Xuan,Li Yunfeng
Artificial cells, nanomedicine, and biotechnology
CircRNA circ_0026344 was previously revealed as a tumour-suppressive gene in colorectal cancer (CRC) progression. The purpose of this research was to investigate the role of circ_0026344 in CRC cells metastasis induced by chemokines. Two human CRC cell lines SW480 and Caco-2 were treated by CCL20 and CXCL8. Cell proliferation, migration/invasion, expression of epithelial-mesenchymal transition (EMT) inducers and the expression of circ_0026344 were measured using sulforhodamine B assay, Transwell chamber, western blot and qRT-PCR, respectively. The effects of circ_0026344 on CRC cells migration/invasion and the expression of EMT inducers were evaluated. Moreover, the downstream miRNA and signalling pathways of circ_0026344 were studied. CCL20 and CXCL8 synergized to facilitate the proliferation, migration and invasion of CRC cells. At the meantime, E-cadherin was downregulated, whereas N-cadherin, Vimentin and Snail were up-regulated by CCL20 and CXCL8 co-stimulation, which was accompanied by the mobilization of PI3K/AKT/ERK signalling. More interestingly, the expression of circ_0026344 was down-regulated by CCL20 and CXCL8 co-stimulation. Silence of circ_0026344 increased the migratory and invasive capacities of CRC cells and increased EMT process as well. Overexpression of circ_0026344 led to a contrary impact. miR-183 was negatively regulated by circ_0026344, and the inhibitory effects of circ_0026344 overexpression on Wnt/β-catenin pathway were reversed when miR-183 was overexpressed. Overexpression of circ_0026344 restrained CRC metastasis and EMT induced by CCL20 and CXCL8 synergistical treatment. miR-183 was a downstream effector of circ_0026344, and the anti-tumour function of circ_0026344 might be involved in the repressed Wnt/β-catenin signalling. Highlights CCL20 and CXCL8 synergize to decrease the expression of circ_0026344; Silence of circ_0026344 promotes CRC cells migration, invasion and EMT process; miR-183 is a downstream effector of circ_0026344.
CXCL8 induces epithelial-mesenchymal transition in colon cancer cells via the PI3K/Akt/NF-κB signaling pathway.
Shen Tao,Yang Zhibin,Cheng Xianshuo,Xiao Youchuan,Yu Kun,Cai Xinyi,Xia Cuifeng,Li Yunfeng
The aim of the present study was to investigate the role of chemokine (C-X-C motif) ligand 8 (CXCL8) in the proliferation, invasiveness and metastasis of colon cancer and its role in the induction of epithelial-mesenchymal transition (EMT) via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor-κB (NF-κB) pathway. The plasmid vector containing CXCL8 cDNA was transfected into LoVo cells using Lipofectamine 2000 reagent. Real-time PCR and western blot analyses were performed to determine expression of CXCL8. MTT growth inhibition, scratch and Transwell invasion assays were conducted to assess cell proliferation, migration and invasiveness of the CXCL8-transfected LoVo cells. Western blot analyses were conducted to measure the levels of phosphorylation of protein in the PI3K/Akt/NF-κB pathway in the CXCL8-transfected LoVo cells. Expression levels of CXCL8 mRNA and protein were significantly increased in the CXCL8-transfected LoVo cells compared with levels in the control and empty-vector cells (P<0.05). Overexpression of CXCL8 increased proliferation of the LoVo cells and significant differences in cell viability were observed 48 h after transfection (P<0.05) and remained significant at 72 and 96 h. CXCL8-transfected LoVo cells had a significantly higher migration rate and doubled invasion. The CXCL8-transfected LoVo cells exhibited an EMT-like phenotype, compared with control and empty-vector cells, with decreased expression of E-cadherin accompanied by increased expression of N-cadherin, vimentin and α-SMA. Overexpression of CXCL8 activated the PI3K/Akt/NF-κB pathway by promoting the phosphorylation of PI3K, Akt and NF-κB. Subcutaneous tumors were generated by subcutaneous injection of LoVo parental cells or CXCL8-transfected LoVo cells in BALB/c nude mice. The tumor growth was more rapid in the CXCL8-transfected group than that noted in the parental cell group. In conclusion, overexpression of CXCL8 induced cell proliferation, migration and invasion of colon cancer LoVo cells. CXCL8 may act through induction of EMT via the PI3K/AKT/NF-κB signaling axis.
Temporal and stochastic control of Staphylococcus aureus biofilm development.
Moormeier Derek E,Bose Jeffrey L,Horswill Alexander R,Bayles Kenneth W
Biofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureus biofilms by observing differential expression of the cid and lrg operons during tower formation. In the present study, we examined early biofilm development and identified two new stages (designated "multiplication" and "exodus") that were associated with changes in matrix composition and a distinct reorganization of the cells as the biofilm matured. The initial attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Interestingly, after 6 h of growth, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyperproliferation of the biofilm and disrupting normal tower development. Immediately prior to the exodus event, S. aureus cells carrying a nuc::gfp promoter fusion demonstrated Sae-dependent expression but only in an apparently random subpopulation of cells. In contrast to the existing model for tower development in S. aureus, the results of this study suggest the presence of a Sae-controlled nuclease-mediated exodus of biofilm cells that is required for the development of tower structures. Furthermore, these studies indicate that the differential expression of nuc during biofilm development is subject to stochastic regulatory mechanisms that are independent of the formation of metabolic microniches. Importance: In this study, we provide a novel view of four early stages of biofilm formation by the human pathogen Staphylococcus aureus. We identified an initial nucleoprotein matrix during biofilm development that is DNase I insensitive until a critical point when a nuclease-mediated exodus of the population is induced prior to tower formation. Unlike the previously described dispersal of cells that occurs after tower development, we found that the mechanism controlling this exodus event is dependent on the Sae regulatory system and independent of Agr. In addition, we revealed that the gene encoding the secreted staphylococcal nuclease was expressed in only a subpopulation of cells, consistent with a model in which biofilms exhibit multicellular characteristics, including the presence of specialized cells and a division of labor that imparts functional consequences to the remainder of the population.
Vision for medicine: Staphylococcus aureus biofilm war and unlocking key's for anti-biofilm drug development.
Mohammed Yasser Hussein Eissa,Manukumar H M,Rakesh K P,Karthik C S,Mallu P,Qin Hua-Li
The Staphylococcus aureus biofilm-associated burden is challenging to the field of medicine to eradicate or avoid it. Even though a number of S. aureus biofilm mechanisms understood and established the possible ways of biofilm formation but, still need to know more and require a development of new therapeutic strategies. In this viewpoint, we discuss the underlining biofilm mechanism, its existing systems as active therapeutic agents and as vehicles to transport drugs to the site of infection. The step-back in drug development is due to the emergence of antibiotic-resistant S. aureus. The understanding of bacteria/biofilms is an aspect that we likewise summarize for possible drug development for future as medicine against resistant S. aureus was viewed.
Biofilm Formation of Staphylococcus aureus under Food Heat Processing Conditions: First Report on CML Production within Biofilm.
Miao Jian,Lin Shiqi,Soteyome Thanapop,Peters Brian M,Li Yanmei,Chen Huishan,Su Jianyu,Li Lin,Li Bing,Xu Zhenbo,Shirtliff Mark E,Harro Janette M
This study aimed to evaluate the Staphylococcus aureus biofilm formation and Nε-carboxymethyl-lysine generation ability under food heat processing conditions including pH (5.0-9.0), temperature (25 °C, 31 °C, 37 °C, 42 °C and 65 °C), NaCl concentration (10%, 15% and 20%, w/v) and glucose concentration (0.5%, 1%, 2%, 3%, 5%, 10%, w/v). S. aureus biofilm genetic character was obtained by PCR detecting atl, ica operon, sasG and agr. Biofilm biomass and metabolic activity were quantified with crystal violet and methyl thiazolyl tetrazolium staining methods. S. aureus biofilm was sensitive to food heat processing conditions with 37 °C, pH 7.0, 2% glucose concentration (w/v) and 10% NaCl concentration (w/v) were favorable conditions. Besides, free and bound Nε-carboxymethyl-lysine level in weak, moderate and strong biofilm were detected by optimized high performance liquid chromatography tandem mass spectrometry. Nε-carboxymethyl-lysine level in S. aureus biofilm possessed a significant gap between strong, moderate and weak biofilm strains. This investigation revealed the biological and chemical hazard of Staphylococcus aureus biofilm to food processing environment.
Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm.
Xu Yuanxi,Jones John E,Yu Haiqing,Yu Qingsong,Christensen Gordon D,Chen Meng,Sun Hongmin
Antimicrobial agents and chemotherapy
Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections.
Effects of lipopeptide carboxymethyl chitosan nanoparticles on Staphylococcus aureus biofilm.
Jiang X H,Zhou W M,He Y Z,Wang Y,Lv B,Wang X M
Journal of biological regulators and homeostatic agents
This study aims to evaluate the effect of lipopeptide carboxymethyl chitosan nanoparticles on Staphylococcus aureus biofilm as part of the development of a new anti-biofilm material. The study had three stages. Firstly, we assessed the Staphylococcus aureus capability to form biofilm and enumerated the number of attached bacteria and free bacteria; secondly, we determined the inhibitory effect of different concentrations of Bacillus natto antimicrobial lipopeptid- carboxymethyl chitosan (BNAP-CMCS) nanoparticles added at different times on biofilm formation capability and the numbers of free bacteria and attached bacteria. Lastly, we tested the scavenging effect of BNAP-CMCS nanoparticles on biofilm formation and number of attached bacteria. The results showed that the amount of attached bacteria quickly increased over time and reached the maximum after 24 h of culture. The BNAP-CMCS nanoparticles had the greatest effect on biofilm inhibition at the concentration of 1 MIC, after 8 h of culture, and the effect was dose-dependent. The BNAP-CMCS nanoparticles had decreased also the numbers of free and attached bacteria in a dose-dependent fashion, after 8 hours of culture. The scavenging effect of BNAP-CMCS nanoparticles on free and attached bacteria was maximum at 6 MIC. In conclusion, lipopeptide carboxymethyl chitosan nanoparticles had a good inhibition and scavenging effect on the formation of Staphylococcus aureus biofilm and the growth of surface-attached bacteria.
Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus.
Vinod Kumar Kirubakaran,Lall Chandan,Raj Ratchagadasse Vimal,Vijayachari Paluru
Microbiology and immunology
It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC-737 showed strong co-aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix-like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin.
Methicillin resistance and the biofilm phenotype in Staphylococcus aureus.
McCarthy Hannah,Rudkin Justine K,Black Nikki S,Gallagher Laura,O'Neill Eoghan,O'Gara James P
Frontiers in cellular and infection microbiology
Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus.
Staphylococcus aureus biofilms: recent developments in biofilm dispersal.
Lister Jessica L,Horswill Alexander R
Frontiers in cellular and infection microbiology
Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and represents a significant burden on the healthcare system. S. aureus attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in the persistence of chronic infections. The formation of a biofilm, and encasement of cells in a polymer-based matrix, decreases the susceptibility to antimicrobials and immune defenses, making these infections difficult to eradicate. During infection, dispersal of cells from the biofilm can result in spread to secondary sites and worsening of the infection. In this review, we discuss the current understanding of the pathways behind biofilm dispersal in S. aureus, with a focus on enzymatic and newly described broad-spectrum dispersal mechanisms. Additionally, we explore potential applications of dispersal in the treatment of biofilm-mediated infections.
Reserpine attenuates biofilm formation and virulence of Staphylococcus aureus.
Parai Debaprasad,Banerjee Malabika,Dey Pia,Mukherjee Samir Kumar
This study investigated the effects of reserpine, the main bioactive compound of Rauwolfia serpentina, on biofilm formation and biofilm-associated virulence factors production in a Gram-positive pathogen, Staphylococcus aureus. Crystal violet assay, MTT assay, Congo red binding, CLSM studies were performed to assess the antibiofilm activity. Molecular docking was performed to explain the possible mode of action, catheter model was used to evaluate its application potential and the combinatorial study was performed in search of an improved therapeutic formulation. Reserpine affected biofilm formation, EPS production, biofilm cell viability and virulence factor production. It could eradicate 72.7% biofilm at ½ × MIC dose and could also stop the metabolic activity of 50.6% bacterial cells in a biofilm. Staphylococcus aureus biofilm- and virulence-regulatory proteins like AgrA, AtlE, Bap, IcaA, SarA and SasG were found to interact with reserpine which might lead to the attenuation of its pathogenicity. Reserpine along with other commercial antibiotics could generate a hightened antibiofilm response, and also eradicated a good percentage of bacterial biofilm from a urinary catheter model. These findings suggested reserpine as a good alternative entity to generate new improved therapeutic formulations.
Alcohol treatment enhances Staphylococcus aureus biofilm development.
Redelman Carly V,Maduakolam Chike,Anderson Gregory G
FEMS immunology and medical microbiology
Staphylococcus aureus forms pathogenic biofilms. Previous studies have indicated that ethanol supplementation during S. aureus biofilm formation results in increased biofilm formation and changes in gene expression. However, the impact of alcohols on preformed S. aureus biofilms has not been studied. In this study, we formed S. aureus biofilms on PVC plastic plates and then treated these preformed biofilms with five different alcohols. We observed that alcohol treatment of preformed S. aureus biofilms led to significant increases in biofilm levels after 24 h of treatment. Many bacteria within these biofilms were found to be alive and metabolically active. Alcohol treatment also resulted in increased transcription of the biofilm-promoting genes icaA and icaD, as well as several antibiotic resistance genes. These results demonstrate that treatment of S. aureus preformed biofilms with alcohols enhances biofilm levels if maintained for extended periods. Thus, alcohols might be of limited usefulness for the eradication of preformed S. aureus biofilms.
Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection.
Ibberson Carolyn B,Parlet Corey P,Kwiecinski Jakub,Crosby Heidi A,Meyerholz David K,Horswill Alexander R
Infection and immunity
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.
Staphylococcus aureus biofilm: a complex developmental organism.
Moormeier Derek E,Bayles Kenneth W
Chronic biofilm-associated infections caused by Staphylococcus aureus often lead to significant increases in morbidity and mortality, particularly when associated with indwelling medical devices. This has triggered a great deal of research attempting to understand the molecular mechanisms that control S. aureus biofilm formation and the basis for the recalcitrance of these multicellular structures to antibiotic therapy. The purpose of this review is to summarize our current understanding of S. aureus biofilm development, focusing on the description of a newly-defined, five-stage model of biofilm development and the mechanisms required for each stage. Importantly, this model includes an alternate view of the processes involved in microcolony formation in S. aureus and suggests that these structures originate as a result of stochastically regulated metabolic heterogeneity and proliferation within a maturing biofilm population, rather than a subtractive process involving the release of cell clusters from a thick, unstructured biofilm. Importantly, it is proposed that this new model of biofilm development involves the genetically programmed generation of metabolically distinct subpopulations of cells, resulting in an overall population that is better able to adapt to rapidly changing environmental conditions.
Staphopains modulate Staphylococcus aureus biofilm integrity.
Mootz Joe M,Malone Cheryl L,Shaw Lindsey N,Horswill Alexander R
Infection and immunity
Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.
Aspartate inhibits Staphylococcus aureus biofilm formation.
Yang Hang,Wang Mengyue,Yu Junping,Wei Hongping
FEMS microbiology letters
Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp.
Propionibacterium acnes biofilm - A sanctuary for Staphylococcus aureus?
Tyner Harmony,Patel Robin
The purpose of this study was to measure the effect of combined culture of Propionibacterium acnes and Staphylococcus aureus on biofilm formation under different oxygen concentrations. We measured planktonic growth and biofilm formation of P. acnes and S. aureus alone and together under aerobic and anaerobic conditions. Both P. acnes and S. aureus grew under anaerobic conditions. When grown under anaerobic conditions, P. acnes with or without S. aureus formed a denser biomass biofilm than did S. aureus alone. Viable S. aureus was recovered from a16-day old combined P. acnes and S. aureus biofilm, but not a monomicrobial S. aureus biofilm.
Inhibition characteristics of biofilm structure of Staphylococcus aureus.
Zhang Liping,Jin Mingxiao,Sun Meng
Cellular and molecular biology (Noisy-le-Grand, France)
Different extracts have different effects on the biofilm structure of Staphylococcus aureus, and the biofilm structure of Staphylococcus aureus will produce different inhibition reactions. In this study, different experimental reagent extracts were used to analyze the inhibition characteristics of Staphylococcus aureus biofilm structure. The inhibition characteristics of bacterial biofilm structure were obtained by using the same bacteria species and the same experimental environment. The results showed that the chloroform extract had a good inhibitory effect on the biofilm structure, which could effectively inhibit the formation of biofilm; the acetic acid extract had an impact on the formation of biofilm, which was destructive to the biofilm; the petroleum ether extract had no effect on the formation of biofilm, that is, it had no inhibitory effect.
The ClpCP Complex Modulates Respiratory Metabolism in Staphylococcus aureus and Is Regulated in a SrrAB-Dependent Manner.
Mashruwala Ameya A,Eilers Brian J,Fuchs Amanda L,Norambuena Javiera,Earle Carly A,van de Guchte Adriana,Tripet Brian P,Copié Valérie,Boyd Jeffrey M
Journal of bacteriology
The taphylococcal espiratory egulator (SrrAB) modulates energy metabolism in Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A Δ strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the Δ strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of was decreased in the Δ mutant, and overexpression of suppressed the puromycin sensitivity of the Δ strain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the Δ and Δ mutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis. Oxygen is used as a substrate to derive energy by the bacterial pathogen during infection; however, can also grow fermentatively in the absence of oxygen. To successfully cause infection, must tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism in We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.
Subinhibitory concentrations of tedizolid potently inhibit extracellular toxin production by methicillin-sensitive and methicillin-resistant Staphylococcus aureus.
Katahira Eva J,Davidson Stephen M,Stevens Dennis L,Bolz Devin D
Journal of medical microbiology
PURPOSE:Potent extracellular toxins including alpha-haemolysin, Panton-Valentine leukocidin (PVL) and toxic-shock syndrome toxin 1 (TSST-1) significantly contribute to Staphylococcus aureus pathogenesis, thus, toxin suppression is a primary focus in treatment of staphylococcal disease. S. aureus maintains complex strategies to regulate toxin expression and previous data have demonstrated that subinhibitory concentrations of beta-lactam antibiotics can adversely increase S. aureus exotoxin production. The current study evaluates the effects of subinhibitory concentrations of tedizolid, a second-generation oxazolidinone derivative, on expression of staphylococcal exotoxins in both methicillin-resistant and methicillin-sensitive S. aureus. METHODOLOGY:S. aureus exotoxin expression levels were compared at 12 and 24 h following treatment with tedizolid, linezolid, nafcillin or vehicle control. RESULTS:Our findings show that the level of antibiotic required to alter toxin production was strain-dependent and corresponds with the quantity of toxin produced, but both tedizolid and linezolid could effectively reduce expression of alpha-haemolysin, PVL and TSST-1 toxin at subinhibitory concentrations. In contrast, nafcillin showed less attenuation and, in some S. aureus strains, led to an increase in toxin expression. Tedizolid consistently inhibited toxin production at a lower overall drug concentration than comparator agents. CONCLUSION:Together, our data support that tedizolid has the potential to improve outcomes of infection due to its superior ability to inhibit S. aureus growth and attenuate exotoxin production.
The good side of inflammation: Staphylococcus aureus proteins SpA and Sbi contribute to proper abscess formation and wound healing during skin and soft tissue infections.
Gonzalez Cintia D,Ledo Camila,Cela Eliana,Stella Inés,Xu Chunliang,Ojeda Diego S,Frenette Paul S,Gómez Marisa I
Biochimica et biophysica acta. Molecular basis of disease
Staphylococcus aureus is the most prominent cause of skin and soft tissue infections (SSTI) worldwide. Mortality associated with invasive SSTI is a major threat to public health considering the incidence of antibiotic resistant isolates in particular methicillin resistant S. aureus both in the hospital (HA-MRSA) and in the community (CA-MRSA). To overcome the increasing difficulties in the clinical management of SSTI due to MRSA, new prophylactic and therapeutic approaches are urgently needed and a preventive vaccine would be welcome. The rational design of an anti-S. aureus vaccine requires a deep knowledge of the role that the different bacterial virulence factors play according to the type of infection. In the present study, using a set of isogenic deficient mutants and their complemented strains we determined that the staphylococcal surface proteins SpA and Sbi play an important role in the induction of inflammatory cytokines and chemokines in the skin during SSTI. SpA and Sbi initiate signaling cascades that lead to the early recruitment of neutrophils, modulate their lifespan in the skin milieu and contribute to proper abscess formation and bacterial eradication. Moreover, the expression of SpA and Sbi appear critical for skin repair and wound healing. Thus, these results indicate that SpA and Sbi can promote immune responses in the skin that are beneficial for the host and therefore, should not be neutralized with vaccine formulations designed to prevent SSTI.
Timing Is Everything: Impact of Naturally Occurring AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction.
Sloan Tim J,Murray Ewan,Yokoyama Maho,Massey Ruth C,Chan Weng C,Bonev Boyan B,Williams Paul
Journal of bacteriology
Mutations in the polymorphic locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for activation via AIP-dependent autoinduction, thus reducing virulence and maintaining in an -downregulated "colonization" mode. Virulence factor expression in is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.
MgrA Governs Adherence, Host Cell Interaction, and Virulence in a Murine Model of Bacteremia Due to Staphylococcus aureus.
Li Liang,Wang Genzhu,Cheung Ambrose,Abdelhady Wessam,Seidl Kati,Xiong Yan Q
The Journal of infectious diseases
BACKGROUND:MgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. METHODS:In vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model. RESULTS:mgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed. CONCLUSIONS:These results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections.
Finding of Agr Phase Variants in Staphylococcus aureus.
Gor Vishal,Takemura Aya J,Nishitani Masami,Higashide Masato,Medrano Romero Veronica,Ohniwa Ryosuke L,Morikawa Kazuya
is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated locus with mechanisms resembling phase variation. The revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.
Disruption of Glycolysis by Nutritional Immunity Activates a Two-Component System That Coordinates a Metabolic and Antihost Response by Staphylococcus aureus.
Párraga Solórzano Paola K,Yao Jiangwei,Rock Charles O,Kehl-Fie Thomas E
During infection, bacteria use two-component signal transduction systems to sense and adapt to the dynamic host environment. Despite critically contributing to infection, the activating signals of most of these regulators remain unknown. This also applies to the ArlRS two-component system, which contributes to virulence by coordinating the production of toxins, adhesins, and a metabolic response that enables the bacterium to overcome host-imposed manganese starvation. Restricting the availability of essential transition metals, a strategy known as nutritional immunity, constitutes a critical defense against infection. In this work, expression analysis revealed that manganese starvation imposed by the immune effector calprotectin or by the absence of glycolytic substrates activates ArlRS. Manganese starvation imposed by calprotectin also activated the ArlRS system even when glycolytic substrates were present. A combination of metabolomics, mutational analysis, and metabolic feeding experiments revealed that ArlRS is activated by alterations in metabolic flux occurring in the latter half of the glycolytic pathway. Moreover, calprotectin was found to induce expression of staphylococcal leukocidins in an ArlRS-dependent manner. These studies indicated that ArlRS is a metabolic sensor that allows to integrate multiple environmental stresses that alter glycolytic flux to coordinate an antihost response and to adapt to manganese starvation. They also established that the latter half of glycolysis represents a checkpoint to monitor metabolic state in Altogether, these findings contribute to understanding how invading pathogens, such as , adapt to the host during infection and suggest the existence of similar mechanisms in other bacterial species. Two-component regulatory systems enable bacteria to adapt to changes in their environment during infection by altering gene expression and coordinating antihost responses. Despite the critical role of two-component systems in bacterial survival and pathogenesis, the activating signals for most of these regulators remain unidentified. This is exemplified by ArlRS, a global regulator that contributes to virulence and to resisting host-mediated restriction of essential nutrients, such as manganese. In this report, we demonstrate that manganese starvation and the absence of glycolytic substrates activate ArlRS. Further investigations revealed that ArlRS is activated when the latter half of glycolysis is disrupted, suggesting that monitors flux through the second half of this pathway. Host-imposed manganese starvation also induced the expression of pore-forming toxins in an ArlRS-dependent manner. Cumulatively, this work reveals that ArlRS acts as a sensor that links nutritional status, cellular metabolism, and virulence regulation.
Interaction in dual species biofilms between Staphylococcus xylosus and Staphylococcus aureus.
Leroy Sabine,Lebert Isabelle,Andant Carine,Talon Régine
International journal of food microbiology
Staphylococcus xylosus, a coagulase-negative Staphylococcus, is frequently isolated from food products of animal origin and used as a starter culture in these products in which it contributes to their flavour, while Staphylococcus aureus, a coagulase-positive bacterium, causes foodborne intoxication and is implicated in a broad diversity of infections in medical sector, notably in nosocomial infections. S. xylosus and S. aureus are both capable of forming a biofilm and share the same ecological niches, thus we explored their interaction in biofilms with a view to limiting the risks associated with S. aureus. Cell-free supernatants of different strains of S. xylosus were able to inhibit the biofilm formation of S. aureus. The S. xylosus C2a strain released into the supernatant a molecule of molecular weight above 30 kDa that is resistant to proteolytic enzymes and inhibits the formation of S. aureus MW2 biofilm, though the mechanism involved has yet to be elucidated. Furthermore, S. xylosus C2a modified the architecture of S. aureus MW2 in co-culture biofilm. Confocal laser scanning microscopy revealed that S. aureus formed a biofilm with a flat and compact structure while in co-culture with S. xylosus the two species formed large juxtaposed aggregates throughout the period of incubation. This architecture made the S. aureus biofilm more susceptible to detachment.