Cranberry-Derived Proanthocyanidins Potentiate ß-Lactam Antibiotics against Resistant Bacteria.

The emergence and spread of extended-spectrum ß-lactamases (ESBLs), metallo-ß-lactamases (MBLs), or variant low-affinity penicillin-binding proteins (PBPs) pose a major threat to our ability to treat bacterial infection using ß-lactam antibiotics. Although combinations of ß-lactamase inhibitors with ß-lactam agents have been clinically successful, there are no MBL inhibitors in current therapeutic use. Furthermore, recent clinical use of new-generation cephalosporins targeting PBP2a, an altered PBP, has led to the emergence of resistance to these antimicrobial agents. Previous work shows that natural polyphenols such as cranberry-extracted proanthocyanidins (cPAC) can potentiate non-ß-lactam antibiotics against Gram-negative bacteria. This study extends beyond previous work by investigating the in vitro effect of cPAC in overcoming ESBL-, MBL-, and PBP2a-mediated ß-lactam resistance. The results show that cPAC exhibit variable potentiation of different ß-lactams against ß-lactam-resistant Enterobacteriaceae clinical isolates as well as ESBL- and MBL-producing E. coli We also discovered that cPAC have broad-spectrum inhibitory properties in vitro on the activity of different classes of ß-lactamases, including CTX-M3 ESBL and IMP-1 MBL. Furthermore, we observe that cPAC selectively potentiate oxacillin and carbenicillin against methicillin-resistant but not methicillin-sensitive staphylococci, suggesting that cPAC also interfere with PBP2a-mediated resistance. This study motivates the need for future work to identify the most bioactive compounds in cPAC and to evaluate their antibiotic-potentiating efficacy in vivoIMPORTANCE The emergence of ß-lactam-resistant Enterobacteriaceae and staphylococci compromises the effectiveness of ß-lactam-based therapy. By acquisition of ESBLs, MBLs, or PBPs, it is highly likely that bacteria may become completely resistant to the most effective ß-lactam agents in the near future. In this study, we described a natural extract rich in proanthocyanidins which exerts adjuvant properties by interfering with two different resistance mechanisms. By their broad-spectrum inhibitory ability, cranberry-extracted proanthocyanidins could have the potential to enhance the effectiveness of existing ß-lactam agents.

Exposure to Freeze-Thaw Conditions Increases Virulence of Pseudomonas aeruginosa to Drosophila melanogaster.

Groundwater contamination by pathogenic bacteria present in land-applied manure poses a threat to public health. In cold climate regions, surface soil layers experience repeated temperature fluctuations around the freezing point known as freeze-thaw (FT) cycles. With global climate change, annual soil FT cycles have increased, and this trend is expected to continue. It is therefore of interest to understand how FT cycles impact soil microbial communities. This study investigates the influence of FT cycles on the growth, culturability, biofilm formation, and virulence of the bacterial opportunistic pathogen Pseudomonas aeruginosa, a ubiquitous bacterium found in soil and water, responsible for infections in immunocompromised hosts. Our findings demonstrate that exposure to FT had no significant effect on growth or culturability of the bacteria. However, FT treatment significantly increased biofilm formation and delayed the onset of swimming motility, factors that are important for the pathogenicity of P. aeruginosa. An in vivo study using a chronic infection model revealed an increase in the virulence of P. aeruginosa after FT exposure. These results suggest that the impact of climate change on natural FT cycles may be affecting the ecology of soil-borne pathogens and host-pathogen interactions in unexpected ways.

The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms.

The significance of extracellular DNA (eDNA) in biofilms was overlooked until researchers added DNAse to a Pseudomonas aeruginosa biofilm and watched the biofilm disappear. Now, a decade later, the widespread importance of eDNA in biofilm formation is undisputed, but detailed knowledge about how it promotes biofilm formation and conveys antimicrobial resistance is only just starting to emerge. In this review, we discuss how eDNA is produced, how it aids bacterial adhesion, secures the structural stability of biofilms and contributes to antimicrobial resistance. The appearance of eDNA in biofilms is no accident: It is produced by active secretion or controlled cell lysis - sometimes linked to competence development. eDNA adsorbs to and extends from the cell surface, promoting adhesion to abiotic surfaces through acid-base interactions. In the biofilm, is it less clear how eDNA interacts with cells and matrix components. A few eDNA-binding biomolecules have been identified, revealing new concepts in biofilm formation. Being anionic, eDNA chelates cations and restricts diffusion of cationic antimicrobials. Furthermore, chelation of Mg(2+) triggers a genetic response that further increases resistance. The multifaceted role of eDNA makes it an attractive target to sensitize biofilms to conventional antimicrobial treatment or development of new strategies to combat biofilms.

Efficiency of CRISPR-Cas9 genetic engineering in Escherichia coli BL21 is impaired by lack of Lon protease.

The efficiency with which E.coli BL21 can be modified using CRISPR-Cas9 genetic engineering is several orders of magnitude lower than that of E. coli W3110. We show that the lack of Lon protease is responsible, and demonstrate that restoration of the Lon protease or knock-out of sulA improves CRISPR-Cas9 engineering efficiency of BL21 to levels comparable to E. coli W3110.

Hydrocarbon bioremediation on Arctic shorelines: Historic perspective and roadway to the future.

Climate change has become one of the greatest concerns of the past few decades. In particular, global warming is a growing threat to the Canadian high Arctic and other polar regions. By the middle of this century, an increase in the annual mean temperature of 1.8 °C-2.7 °C for the Canadian North is predicted. Rising temperatures lead to a significant decrease of the sea ice area covered in the Northwest Passage. As a consequence, a surge of maritime activity in that region increases the risk of hydrocarbon pollution due to accidental fuel spills. In this review, we focus on bioremediation approaches on Arctic shorelines. We summarize historical experimental spill studies conducted at Svalbard, Baffin Island, and the Kerguelen Archipelago, and review contemporary studies that used modern omics techniques in various environments. We discuss how omics approaches can facilitate our understanding of Arctic shoreline bioremediation and identify promising research areas that should be further explored. We conclude that specific environmental conditions strongly alter bioremediation outcomes in Arctic environments and future studies must therefore focus on correlating these diverse parameters with the efficacy of hydrocarbon biodegradation.

Hydrocarbon biodegradation potential of microbial communities from high Arctic beaches in Canada's Northwest Passage.

Sea ice loss is opening shipping routes in Canada's Northwest Passage, increasing the risk of an oil spill. Harnessing the capabilities of endemic microorganisms to degrade oil may be an effective remediation strategy for contaminated shorelines; however, limited data exists along Canada's Northwest Passage. In this study, hydrocarbon biodegradation potential of microbial communities from eight high Arctic beaches was assessed. Across high Arctic beaches, community composition was distinct, potential hydrocarbon-degrading genera were detected and microbial communities were able to degrade hydrocarbons (hexadecane, naphthalene, and alkanes) at low temperature (4 °C). Hexadecane and naphthalene biodegradation were stimulated by nutrients, but nutrients had little effect on Ultra Low Sulfur Fuel Oil biodegradation. Oiled microcosms showed a significant enrichment of Pseudomonas and Rhodococcus. Nutrient-amended microcosms showed increased abundances of key hydrocarbon biodegradation genes (alkB and CYP153). Ultimately, this work provides insight into hydrocarbon biodegradation on Arctic shorelines and oil-spill remediation in Canada's Northwest Passage.

Biofilm formation by marine bacteria is impacted by concentration and surface functionalization of polystyrene nanoparticles in a species-specific manner.

The world's oceans are becoming increasingly polluted by plastic waste. In the marine environment, larger plastic pieces may degrade into nanoscale (<100 nm in at least one dimension) plastic particles due to natural weathering effects. We observe that the presence of 20 nm plastic nanoparticles at concentrations below 200 ppm had no impact on planktonic growth of a panel of heterotrophic marine bacteria. However, the presence of plastic nanoparticles significantly impacted the formation of biofilms in a species-specific manner. While carboxylated nanoparticles increased the amount of biofilm formed by several species, amidine-functionalized nanoparticles decreased the amount of biofilm of many but not all bacteria. Further experiments suggested that the aggregation dynamics of bacteria and nanoparticles were strongly impacted by the surface properties of the nanoparticles. The community structure of an artificially constructed community of marine bacteria was significantly altered by exposure to plastic nanoparticles, with differently functionalized nanoparticles selecting for unique and reproducible community abundance patterns. These results suggest that surface properties and concentration of plastic nanoparticles, as well as species interactions, are important factors determining how plastic nanoparticles impact biofilm formation by marine bacteria.

Antimicrobial Hierarchically Porous Graphene Oxide Sponges for Water Treatment.

Water treatment technologies based on graphene oxide (GO) sponges show promise due to their high surface area and versatile chemistry, yielding an excellent adsorption affinity for different contaminants. However, the bacteria removal capacity and the intrinsic antimicrobial properties of GO sponges are not well understood. While GO has been successfully functionalized with antibiotics or metal biocides, these antimicrobials present cytotoxicity concerns. Natural antimicrobial agents such as antimicrobial enzymes, peptides, and polymers hold promise in this respect due to their relatively low cost, biocompatibility, and ability to readily functionalize GO by covalent bond formation with oxygen-containing functional groups. In this work, the antimicrobial enzyme lysozyme, antimicrobial peptide nisin, and antimicrobial polyamide ε-poly-l-lysine were used to covalently functionalize the surface of a hierarchically porous GO sponge. The antimicrobial activity of the functionalized material was demonstrated against two model organisms: the Gram-positive B. subtilis and Gram-negative E. coli. The performance of the porous material in a simulated filtration context was evaluated using packed column experiments, and an improved bacterial retention of both strains by the functionalized sponge was demonstrated. Furthermore, samples of spent sponge after filtration were evaluated with a membrane integrity assay demonstrating antimicrobial activity in a continuous flow mode.

Proanthocyanidin Interferes with Intrinsic Antibiotic Resistance Mechanisms of Gram-Negative Bacteria.

Antibiotic resistance is spreading at an alarming rate among pathogenic bacteria in both medicine and agriculture. Interfering with the intrinsic resistance mechanisms displayed by pathogenic bacteria has the potential to make antibiotics more effective and decrease the spread of acquired antibiotic resistance. Here, it is demonstrated that cranberry proanthocyanidin (cPAC) prevents the evolution of resistance to tetracycline in Escherichia coli and Pseudomonas aeruginosa, rescues antibiotic efficacy against antibiotic-exposed cells, and represses biofilm formation. It is shown that cPAC has a potentiating effect, both in vitro and in vivo, on a broad range of antibiotic classes against pathogenic E. coli, Proteus mirabilis, and P. aeruginosa. Evidence that cPAC acts by repressing two antibiotic resistance mechanisms, selective membrane permeability and multidrug efflux pumps, is presented. Failure of cPAC to potentiate antibiotics against efflux pump-defective mutants demonstrates that efflux interference is essential for potentiation. The use of cPAC to potentiate antibiotics and mitigate the development of resistance could improve treatment outcomes and help combat the growing threat of antibiotic resistance.

Culture-Dependent Bioprospecting of Bacterial Isolates From the Canadian High Arctic Displaying Antibacterial Activity.

The goal of this study was to isolate, screen, and characterize Arctic microbial isolates from Expedition Fjord, Axel Heiberg Island, Nunavut, Canada capable of inhibiting the growth of foodborne and clinically relevant pathogens. Arctic bacteria were isolated from twelve different high Arctic habitats pertaining to active layer permafrost soil, saline spring sediments, lake sediments, and endoliths. This was achieved using (1) the cryo-iPlate, an innovative in situ cultivation device within active layer permafrost soil and (2) bulk plating of Arctic samples by undergraduate students that applied standard culturing methods. To mitigate the possibility of identifying isolates with already-known antibacterial activities, a cell-based dereplication platform was used. Ten out of the twelve Arctic habitats tested were found to yield cold-adapted isolates with antibacterial activity. Eight cold-adapted Arctic isolates were identified with the ability to inhibit the entire dereplication platform, suggesting the possibility of new mechanisms of action. Two promising isolates, initially cultured from perennial saline spring sediments and from active layer permafrost soil (Paenibacillus sp. GHS.8.NWYW.5 and Pseudomonas sp. AALPS.10.MNAAK.13, respectively), displayed antibacterial activity against foodborne and clinically relevant pathogens. Paenibacillus sp. GHS.8.NWYW.5 was capable of inhibiting methicillin resistant and susceptible Staphylococcus aureus (MRSA and MSSA), Listeria monocytogenes, Salmonella enterica and Escherichia coli O157:H7. Pseudomonas sp. AALPS.10.MNAAK.13 was observed to have antagonistic activity against MRSA, MSSA, Acinetobacter baumanii, Enterococcus faecium, and Enterococcus faecalis. After whole genome sequencing and mining, the genome of Paenibacillus sp. GHS.8.NWYW.5 was found to contain seven putative secondary metabolite biosynthetic gene clusters that displayed low homology (<50% coverage, <30% identity, and e-values > 0) to clusters identified within the genome of the type strain pertaining to the same species. These findings suggest that cold-adapted Arctic microbes may be a promising source of novel secondary metabolites for potential use in both industrial and medical settings.

Anodized Aluminum with Nanoholes Impregnated with Quaternary Ammonium Compounds Can Kill Pathogenic Bacteria within Seconds of Contact.

Bacterial contamination of surfaces results in the spread of pathogens in public spaces such as hospitals and public transport. The development of antibacterial surfaces that rapidly kill bacteria is therefore highly desirable. Here, we investigate the antibacterial efficacy of a novel anodized aluminum surface featuring nanoholes impregnated with quaternary ammonium compounds, referred to as A3S. The antimicrobial activity of A3S was assessed using both Gram-positive and Gram-negative bacteria in a novel assay which simulates pathogen transfer from a contaminated "finger" to a clean finger in a real-world scenario. Enumeration of colony-forming units shows that the number of viable bacteria on the second "finger" contacting A3S is significantly reduced compared to a control surface. Furthermore, bacterial contact with the A3S material results in compromised cell membranes in less than 1 min, and a kill zone assay shows that an exposure time as short as 5 s is sufficient to kill pathogenic bacteria. The rapid antimicrobial action of A3S was particularly evident against Gram-positive bacteria, that account for more than 70% of nosocomial infections. Taken together, these findings demonstrate that A3S is a promising candidate for the fabrication of antibacterial surfaces that can be used in a wide range of clinical and commercial applications to stop the spread of harmful bacteria.

Developing Antibacterial Nanocrystalline Cellulose Using Natural Antibacterial Agents.

We used hairy nanocrystalline cellulose functionalized with aldehyde groups, otherwise known as sterically stabilized nanocrystalline cellulose (SNCC), to facilitate the attachment of the antibacterial agents lysozyme and nisin. Immobilization was achieved using a simple, green process that does not require any linker or activator. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy analyses showed successful attachment of both nisin and lysozyme onto the SNCC. The efficacy of the conjugated nanocellulose against the model bacteria Bacillus subtilis and Staphylococcus aureus was tested in terms of bacterial growth, cell viability, and biofilm formation/removal. The results show that the minimum inhibitory concentration of the conjugated nanocellulose is higher than that of lysozyme and nisin in free form, which was expected given that immobilization reduces the possible spatial orientations of these proteins. We observed that free nisin is not active against S. aureus after 24 h of exposure due to either deactivation of free nisin or development of resistance in S. aureus against free nisin. Interestingly, we did not observe this phenomenon when the bacteria were exposed to antibacterials immobilized on nanocellulose, suggesting that immobilization of antibacterial agents onto SNCC effectively retains their activity over long time periods. We suggest that antibacterial SNCC is a promising candidate for the development of antibacterial wound dressings.

A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation.

Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 +  transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus.

Draft Genome Sequence of Bacillus sp. FMQ74, a Dairy-Contaminating Isolate from Raw Milk.

Representatives of the genus Bacillus are common milk contaminants that cause spoilage and flavor alterations of dairy products. Bacillus sp. FMQ74 was isolated from raw milk on a Danish dairy farm. To elucidate the genomic basis of this strain's survival in the dairy industry, a high-quality draft genome was produced.

Extracellular DNA as a target for biofilm control.

Bacterial biofilms endure high concentrations of biocides, and new strategies for biofilm control must therefore replace or complement the use of antibiotics, for example, by targeting the extracellular matrix to cause dispersal or increased antimicrobial susceptibility. Extracellular DNA (eDNA) is a matrix component of most biofilms, and is therefore an attractive target. Enzymatic degradation of eDNA can prevent, disperse, or sensitize biofilm to antimicrobials, but cheaper production is required to realize large-scale application. Replacing mammalian DNase with bacterial nucleases could offer a path to lower production costs. Alternatively, eDNA could be targeted by disrupting its interactions with other matrix components. As new knowledge about eDNA-binding matrix components comes to light, exciting opportunities for targeting the biofilm matrix via eDNA are emerging.

DNase-Sensitive and -Resistant Modes of Biofilm Formation by Listeria monocytogenes.

Listeria monocytogenes is able to form biofilms on various surfaces and this ability is thought to contribute to persistence in the environment and on contact surfaces in the food industry. Extracellular DNA (eDNA) is a component of the biofilm matrix of many bacterial species and was shown to play a role in biofilm establishment of L. monocytogenes. In the present study, the effect of DNaseI treatment on biofilm formation of L. monocytogenes EGD-e was investigated under static and dynamic conditions in normal or diluted complex medium at different temperatures. Biofilm formation was quantified by crystal violet staining or visualized by confocal laser scanning microscopy. Biomass of surface-attached L. monocytogenes varies depending on temperature and dilution of media. Interestingly, L. monocytogenes EGD-e forms DNase-sensitive biofilms in diluted medium whereas in full strength medium DNaseI treatment had no effect. In line with these observations, eDNA is present in the matrix of biofilms grown in diluted but not full strength medium and supernatants of biofilms grown in diluted medium contain chromosomal DNA. The DNase-sensitive phenotype could be clearly linked to reduced ionic strength in the environment since dilution of medium in PBS or saline abolished DNase sensitivity. Several other but not all species of the genus Listeria display DNase-sensitive and -resistant modes of biofilm formation. These results indicate that L. monocytogenes biofilms are DNase-sensitive especially at low ionic strength, which might favor bacterial lysis and release of chromosomal DNA. Since low nutrient concentrations with increased osmotic pressure are conditions frequently found in food processing environments, DNaseI treatment represents an option to prevent or remove Listeria biofilms in industrial settings.

Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms.

Here we determine an optimal technique for the visualization of extracellular DNA in bacterial biofilms using the fluorescent eDNA stain TOTO-1 and the counterstain SYTO 60. This technique allows for more sensitive eDNA visualization than other fluorescent staining methods currently in use.