Investigating the Link Between Imipenem Resistance and Biofilm Formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC?≤?2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC?≥?8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.     

Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation

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

Tannins Possessing Bacteriostatic Effect Impair Pseudomonas aeruginosa Adhesion and Biofilm Formation

Plants produce many compounds that are biologically active, either as part of their normal program of growth and development or in response to pathogen attack or stress. Traditionally, Anadenanthera colubrina, Commiphora leptophloeos and Myracrodruon urundeuva have been used by communities in the Brazilian Caatinga to treat several infectious diseases. The ability to impair bacterial adhesion represents an ideal strategy to combat bacterial pathogenesis, because of its importance in the early stages of the infectious process; thus, the search for anti-adherent compounds in plants is a very promising alternative. This study investigated the ability of stem-bark extracts from these three species to control the growth and prevent biofilm formation of Pseudomonas aeruginosa, an important opportunistic pathogen that adheres to surfaces and forms protective biofilms. A kinetic study (0–72 h) demonstrated that the growth of extract-treated bacteria was inhibited up to 9 h after incubation, suggesting a bacteriostatic activity. Transmission electron microscopy and fluorescence microscopy showed both viable and nonviable cells, indicating bacterial membrane damage; crystal violet assay and scanning electron microscopy demonstrated that treatment strongly inhibited biofilm formation during 6 and 24 h and that matrix production remained impaired even after growth was restored, at 24 and 48 h of incubation. Herein, we propose that the identified (condensed and hydrolyzable) tannins are able to inhibit biofilm formation via bacteriostatic properties, damaging the bacterial membrane and hindering matrix production. Our findings demonstrate the importance of this abundant class of Natural Products in higher plants against one of the most challenging issues in the hospital setting: biofilm resilience.     

Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation by L. rhamnosus GG.     

Properties of the Probiotic Strain Lactobacillus plantarum 8-RA-3 Grown in a Biofilm by Solid Substrate Cultivation Method

The biofilm formation took place in 48?h within the solid substrate cultivation of Lactobacillus plantarum 8-RA-3 strain on the wheat bran saturated with the MRS medium. The drying of the bran fermented by lactobacilli resulted in a decrease in the number of colony-forming units (CFU) from 23.0?×?10⁸ to 6.9?×?10⁵?CFU/g in daily samples and to less than 10⁴?CFU/g in 2- and 3-day samples. However, according to the fluorescence-based live/dead assay data, more than 40?% of the non-cultured bacteria were viable. As a result of mice kept on a diet with the introduction of bran fermented by Lact. plantarum 8-RA-3 for 72?h into the fodder, a recovery of normal level of intestinal lactobacilli, inhibited by administration of antibiotic was noted. The strain genetically identical to the Lact. plantarum 8-RA-3 was isolated from the feces of these mice. The results indicate that solid substrate cultivated Lact. plantarum 8-RA-3 strain formed a biofilm. Once dried and transferred into a non-cultured state, biofilm cells retained its viability and biological activity.     

Anti-infective properties of Lactobacillus fermentum against Staphylococcus aureus and Pseudomonas aeruginosa

Surgical wounds and implant-associated Staphylococcus aureus and Pseudomonas aeruginosa infections are often difficult to treat because of limited susceptibility of several of these strains to conventional antibiotics. As a result, there is a constant need for new alternative drugs. The aim of this study was to investigate the antimicrobial properties of Lactobacillus fermentum, a probiotic bacterium, which we have isolated from colonic biopsies. The inhibition of S. aureus and P. aeruginosa growth was evaluated by coincubating with L. fermentum strains. Growth inhibition was tested for several of their clinical isolates using agar well diffusion assays. For biofilm assay S. aureus and P. aeruginosa were grown on the glass slides and in 96-well plates in presence of 2.5 μg/ml culture filtrate of L. fermentum. Biofilms were photographed using confocal microscope or stained with 0.1% crystal violet. Reduction in the cytotoxicity of S. aureus and P. aeruginosa was observed in presence of 2.5 μg/ml L. fermentum-spent media. Using in vitroexperiments, we showed that L. fermentum-secreted compound(s) inhibits the growth, cytotoxicity and biofilm formation of several S. aureus and P. aeruginosa strains. Compound(s) present in the culture supernatant of L. fermentum may have promising applications in treating hospital-acquired infections.     

铜绿假单胞菌Arr基因突变对生物膜和绿脓菌素合成的影响

为了研究铜绿假单胞菌Arr基因对生物膜和绿脓菌素合成的影响,采用抗庆大霉素基因序列(Gentamycin resistance cassette,aacC1)插入失活的策略构建了铜绿假单胞菌Arr基因突变株PA-AG,通过96孔板静止培养、结晶紫染色的方法检测其生物膜的形成量,利用抽提的方法检测绿脓菌素的合成量。结果在KMB或LB培养基中,突变株PA-AG形成生物膜的量均有所减少,野生株约是突变株的2倍,然而突变株合成绿脓菌素的能力却明显加强,约为野生株的2.5倍。由此推测,铜绿假单胞菌Arr基因在一定程度上促进了生物膜的形成,抑制了绿脓菌素的合成。     

Protective effect of lipoteichoic acid from Lactobacillus casei and Lactobacillus fermentum against Pseudomonas aeruginosa in mice

Lipoteichoic acid (LTA) from Lactobacillus casei YIT 9018 or Lactobacillus fermentum YIT 0159 augmented the resistance of C57BL/6 mice to infection with Pseudomonas aeruginosa, but conferred no resistance to Listeria monocytogenes. It is suggested that LTA was unable to activate macrophages.     

Pseudomonas aeruginosa Utilizes Host-Derived Itaconate to Redirect Its Metabolism to Promote Biofilm Formation

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The Mechanism of Formation of Pseudomonas aeruginosa Biofilm,a Type of Structured Population

The paper is an attempt to analyze and generalize molecular and cell biology data on the formation of polysaccharide matrix–based biofilms. The conception of biofilms as structured populations sharing the characteristics of uni- and multicellular organisms and population is proposed.     

Biofilm inhibition and anti-quorum sensing activity of phytosynthesized silver nanoparticles against the nosocomial pathogen Pseudomonas aeruginosa

Quorum sensing (QS), the communication signaling network, regulates biofilm formation and several virulence factors in Pseudomonas aeruginosa PAO1, a nosocomial opportunistic pathogen. QS is considered to be a challenging target for compounds antagonistic to virulent factors. Biologically synthesized silver nanoparticles (AgNPs) are reported as anti-QS and anti-biofilm drugs against bacterial infections. The present study reports on the synthesis and characterization of Piper betle (Pb) mediated AgNPs (Pb-AgNPs). The anti-QS activity of Pb-AgNPs against Chromobacterium violaceum and the potential effect of Pb-AgNPs on QS-regulated phenotypes in PAO1 were studied. FTIR analysis exhibited that Pb-AgNPs had been capped by phytochemical constituents of Pb. Eugenol is one of the active phenolic phytochemicals in Pb leaves, therefore molecular docking of eugenol-conjugated AgNPs on QS regulator proteins (LasR, LasI and MvfR) was performed. Eugenol-conjugated AgNPs showed considerable binding interactions with QS-associated proteins. These results provide novel insights into the development of phytochemically conjugated nanoparticles as promising anti-infective candidates.     

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

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

Lactonase-expressing Lactobacillus plantarum NC8 attenuates the virulence factors of multiple drug resistant Pseudomonas aeruginosa in co-culturing environment

Pseudomonas aeruginosa possesses an arcade of both cell-associated and extracellular cytotoxic virulence factors which are regulated by a multi-component quorum sensing system. Many research studies report success of lactonase in combating the pathogenicity of P. aeruginosa but delivery of lactonase remains a challenge. The present study aims at developing a delivery vehicle for lactonase. Lactobacillus plantarum NC8 was used as host for aiiA (Bacillus thuringiensis 4A3 lactonase gene) using pSIP409 expression vector. pSIP409: aiiA construct was stably maintained in L. plantarum NC8. Co-culturing of multi-drug resistant (MDR) clinical isolates of P. aeruginosa and PAO1 with recombinant L. plantarum NC8 led to significant reduction (p < 0.001) in extracellular virulence factors like pyocyanin, protease, elastase and rhamnolipids in P. aeruginosa and also showed significant reduction in adhesion of P. aeruginosa strains to uroepithelial cells in vitro. This study shows the heterologous expression of AiiA lactonase in L. plantarum NC8. Co-culturing of lactonase expressing L. plantarum NC8 with MDR P. aeruginosa strains led to attenuation of their virulence significantly. These results underscore the potential application of recombinant L. plantarum NC8 with anti-quorum sensing properties to control infections caused by multidrug resistant P. aeruginosa.     

铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌的抑菌活性研究

目的观察铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcusaureus,MRSA)的体外抑菌活性。方法用交叉划线接种方法进行铜绿假单胞菌对32株耐甲氧西林金葡菌的体外抗菌活性的测定。结果铜绿假单胞菌抗菌物质对MRSA的体外抑菌活性良好,产生色素的菌株的抗菌活性最好,15株铜绿假单胞菌中,7株产蓝绿色色素的铜绿假单胞菌,对MRSA的抑制率均达到了100%,平均抑菌带的宽度为37.7 mm。结论铜绿假单胞菌抗菌物质对32株MRSA具有较强的抗菌活性,无疑对MRSA感染的抗菌药物研制方面开辟了一条新的途径。这是国内的首次研究报道。     

Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

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

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

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa

Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.     

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

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