Novel bioactive peptides from PD-L1/2, a type 1 ribosome inactivating protein from Phytolacca dioica L. Evaluation of their antimicrobial properties and anti-biofilm activities

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.     

Extracts of seaweeds as potential inhibitors of quorum sensing and bacterial growth

Macroalgae are an important source of antimicrobial compounds. However, it is unclear if these compounds are produced by the algae themselves, by their associated bacteria, or by both. The main aim of this study was to investigate the potential of macroalgae and their associated microorganisms to inhibit bacterial quorum sensing (QS) and growth. Before extraction, half of the algal specimens were treated with 30% ethanol to remove surface associated bacteria. Canistrocarpus cervicornis extracts were able to inhibit QS of the reporter Chromobacterium violaceum CV017, where extracts with associated bacteria were more efficient than those without bacteria. However, not all algal extracts that inhibited QS of CV017 were able to inhibit bacterial attachment of Pseudomonas aeruginosas PA01, showing specific activity of algal metabolites. Only 58% of the extracts showed antibacterial activity against eight marine fouling and pathogenic bacterial strains tested. Our data suggests that algae and their associated microbiota are important sources of antimicrobial compounds which potentially can be used in future biotechnological applications.     

Mechanisms of biofilm resistance to antimicrobial agents

Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The physical and/or chemical structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.     

Resistance to tetracycline, macrolide-lincosamide-streptogramin, trimethoprim, and sulfonamide drug classes

The discovery and use of antimicrobial agents in the last 50 yr has been one of medicine’s greatest achievements. These agents have reduced morbidity and mortality of humans and animals and have directly contributed to human’s increased life span. However, bacteria are becoming increasingly resistant to these agents by mutations, which alter existing bacterial proteins, and/or acquisition of new genes, which provide new proteins. The latter are often associated with mobile elements that can be exchanged quickly across bacterial populations and may carry multiple antibiotic genes fo resistance. In some case, virulence factors are also found on these same mobile elements. There is mounting evidence that antimicrobial use in agriculture, both plant and animal, and for environmental purposes does influence the antimicrobial resistant development in bacteria important in humans and in reverse. In this article, we will examine the genes which confer resistance to tetracycline, macrolide-lincosamide-streptogramin (MLS), trimethoprim, and sulfonamide.     

Antimicrobial Peptides and their Potential as Oral Therapeutic Agents

Dental caries (tooth decay) and periodontal diseases are the most prevalent bacterial infectious diseases of mankind, together affecting almost the entire population of the world. Both diseases are caused by oral bacteria that exist as components of a polymicrobial biofilm, known as dental plaque, on the tooth surface. The control of specific types of bacteria and/or total numbers of bacteria in dental plaque could lead to prevention or resolution of disease. Antimicrobial peptides isolated from a wide range of natural sources have been known for over 30 years yet little progress had been made in the therapeutic application of these peptides. This is due in part to the characteristics, including susceptibility to proteolysis, of the cationic amphipathic antimicrobial peptides that form the majority of peptides discovered to date. Bovine milk is a readily available source of a range of bioactive peptides. We have isolated and characterized a novel anionic antimicrobial peptide, Kappacin, from bovine milk. Antibacterial activity of the peptide is increased when it is complexed with zinc ions. We have demonstrated that a Kappacin:Zn²⁺ preparation is able to suppress the growth of oral cariogenic bacteria in a biofilm. The Kappacin:Zn²⁺ antibacterial complex may have potential as an additive to oral care products and other delivery vehicles for the control of oral disease.     

Polypropylene with embedded copper metal or copper oxide nanoparticles as a novel plastic antimicrobial agent

Aims: To develop novel polypropylene composite materials with antimicrobial activity by adding different types of copper nanoparticles. Methods and Results: Copper metal (CuP) and copper oxide nanoparticles (CuOP) were embedded in a polypropylene (PP) matrix. These composites present strong antimicrobial behaviour against E. coli that depends on the contact time between the sample and the bacteria. After just 4 h of contact, these samples are able to kill more than 95% of the bacteria. CuOP fillers are much more effective eliminating bacteria than CuP fillers, showing that the antimicrobial property further depends on the type of copper particle. Cu²⁺ released from the bulk of the composite is responsible for this behaviour. Moreover, PP/CuOP composites present a higher release rate than PP/CuP composites in a short time, explaining the antimicrobial tendency. Conclusions: Polypropylene composites based on copper nanoparticles can kill E. coli bacteria depending on the release rate of Cu²⁺ from the bulk of the material. CuOP are more effective as antimicrobial filler than CuP. Significance and Impact of the Study: Our findings open up novel applications of these ion‐copper‐delivery plastic materials based on PP with embedded copper nanoparticles with great potential as antimicrobial agents.     

Transposon mutagenesis identifies genes which control antimicrobial drug tolerance in stationary-phase Escherichia coli

Tolerance to antimicrobial agents is a universal phenomenon in bacteria which are no longer multiplying or whose growth rate slows. Since slowly multiplying bacteria occur in clinical infections, extended periods of antimicrobial chemotherapy are needed to eradicate these organisms and to achieve cure. In this study, the molecular basis of antibiotic tolerance was investigated using transposon mutagenesis. We screened 5000 Escherichia coli Tn10Cam mutants for reduction of kanamycin tolerance in late stationary phase and found that 4935 mutants were able to grow to late stationary phase. Reduced tolerance was observed in nine mutants which became sensitive to killing by kanamycin. The mutant KS639 was the most sensitive one to kanamycin, and its genome was disrupted in an intergenic region which lies between aldB and yiaW open reading frames. This mutant showed increased sensitivity not only to kanamycin but also to gentamicin, ciprofloxacin and rifampicin. Reduced tolerance of KS639 to kanamycin was also observed in a murine thigh infection model. P1 transduction to the wild type strains confirmed that the intergenic region was responsible for the tolerance of the bacterium to antibiotics. Using PCR-directed one-step gene replacement, we inactivated the genes aldB, yiaW and yiaV. We also deleted the intergenic region. There was no difference in kanamycin tolerance between each mutant (DeltaaldB, DeltayiaW and DeltayiaV) and the parental strain. But the mutant lacking the intergenic region showed reduced tolerance to kanamycin. These data suggest that the intergenic region between aldB and yiaW genes may be involved in tolerance to antimicrobial agents in E. coli. Furthermore, they show that it is important in murine infection during antibiotic treatment and lead to a faster kill of the mutant bacteria.     

Metabolic adaptation of human pathogenic and related nonpathogenic bacteria to extra- and intracellular habitats

Most bacteria pathogenic for humans have closely related nonpathogenic counterparts that live as saprophytes, commensals or even symbionts (mutualists) in similar or different habitats. The knowledge of how these bacteria adapt their metabolism to the preferred habitats is critical for our understanding of pathogenesis, commensalism and symbiosis, and - in the case of bacterial pathogens - could help to identify targets for new antimicrobial agents. The focus of this review is on the metabolic potentials and adaptations of three different groups of human extra- and intracellular bacterial pathogens and their nonpathogenic relatives. All bacteria selected have the potential to reach the interior of mammalian host cells. However, their ability to replicate intracellularly differs significantly. The question therefore arises whether there are specific metabolic requirements that support stable intracellular replication. Furthermore, we discuss - whenever relevant data for the pathogenic representatives are available - the possible effect of the metabolism on the expression of virulence genes.     

The role of anaerobic bacteria in sinusitis

The normal oropharyngeal flora contained aerobic and anaerobic bacteria that can cause respiratory infections including sinusitis. Some of these bacteria can interfere with the growth of potential pathogens and may play a role in preventing infections. Anaerobic bacteria emerge as pathogens as the infection becomes chronic. This may be the result of the selective pressure of antimicrobial agents that enable resistant anaerobic organisms to survive, and from the development over time of conditions appropriate for anaerobic growth, which include the reduction in oxygen tension and an increase in acidity within the sinus cavity. Anaerobes were isolated in acute maxillary sinusitis of odontogenic origin and in over half of the patients with chronic sinusitis whenever proper techniques for their cultivation were employed. These organisms were also recovered in acute sinusitis that was associated with dental infections. The predominant isolates were pigmented Prevotella and Porphyromonas, Fusobacterium and Peptostreptococcus spp.     

Sensitivity of environmental microorganisms to antimicrobial agents.

The sensitivity of different microorganisms, considered as typical representatives of the microflora of soil and water, was established to evaluate the influence of the nonmedical use of antimicrobial agents on bacterial ecology. Only seven strains, six chemoorganotrophs and one chemolithotroph, could be considered as relatively sensitive to the 21 antimicrobial compounds tested. The other 29 microorganisms may be regarded as resistant to most antimicrobial agents. Streptomyces were sensitive to high concentrations of active substances. Broad-spectrum antibiotics showed an effect on environmental bacteria similar to that on human pathogens. Cephalothin stimulated the growth of a Chlorella sp. From these experiments, it appears that spilled antimicrobial agents have little chance of causing an alteration in the microbial ecology.     

De-Novo Design of Antimicrobial Peptides for Plant Protection

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents.     

Collective motion of surfactant-producing bacteria imparts superdiffusivity to their upper surface

Swarming bacteria move on agar surfaces in groups, using flagella as motive organelles. Motility depends critically on surface wetness, which is enabled by osmotic agents and surfactants secreted by the bacteria. In a recent study, the upper surface of an Escherichia coli swarm was found to be stationary, as determined from the motion of MgO particles deposited on the swarm. This led to the remarkable conclusion that the bacteria move between two stationary surfaces—the agar gel below and the liquid/air interface above. That study suggested that secreted surfactants may contribute to immobilizing the upper surface of a swarm. Here, we test this proposition using two robust surfactant-producing bacteria. We find antithetically that the upper surfaces of both these swarms are mobile, showing a superdiffusive behavior in swarms with stronger surfactant activity. Superdiffusive behavior was not observed on the surface of a drop of bacterial culture, on bacteria-free culture supernatant, or on nonswarming surfactant-producer colonies, which suggests that superdiffusion is an emergent property resulting from the interaction of the collective motion of the bacteria within the swarm with the surfactant layer above. Swarming not only allows bacteria to forage for food, but also confers protective advantages against antimicrobial agents. Our results are therefore relevant to superdiffusive strategies in biological foraging and survival.     

Two Plant Puroindolines Colocalize in Wheat Seed and <Emphasis Type="Italic">in vitro</Emphasis> Synergistically Fight Against Pathogens

Puroindolines, for years largely investigated for their involvement in wheat kernel hardness, have recently attracted attention thanks to their possible role as antimicrobial proteins. With the aim to enhance our knowledge of these proteins we studied their localization in the kernel, and their antimicrobial activity in vitro against six different bacterial strains. Immunolocalization showed that both the PINs are strongly concentrated in the aleurone layer, but also highly present in the endosperm. Interestingly we observed that puroindolines not only have the same spatial distribution in the kernel, they are also always found co-localized. Their co-localization suggests that they could cooperate in defending the plant against pathogens. We therefore tested antimicrobial activity of PINA and PINB, and a putative synergism between these proteins. The results showed that the two polypeptides can in vitro inhibit growth of all the bacteria tested; furthermore when combined together they are able to enhance each other’s toxicity. In view of their antimicrobial activity and of their natural presence in Triticum aestivum wheat flour, puroindolines look promising antibacterial agents and thus deserve further studies aimed at establishing their possible future applications in fields of food and health care. Since PINs were still detectable in bakery products, these proteins may be promising tools in investigating natural ways of food preservation.     

Lethal photosensitisation of oral bacteria and its potential application in the photodynamic therapy of oral infections.

Chemical antibacterial agents are increasingly being used in prophylactic and therapeutic regimes for dental plaque-related diseases, which are among the most common human infections. As these agents are difficult to maintain at a therapeutic concentration in the oral cavity and can be rendered ineffective by resistance development in the target organisms, there is a need to develop alternative antimicrobial approaches. Bacteria and other microbes can be sensitised to light through prior treatment with a chemical photosensitising agent. Lethal photosensitisation of a wide range of bacteria responsible for caries, periodontal diseases and root canal infections has been demonstrated using red light in conjunction with a number of photosensitisers, including Toluidine Blue, phthalocyanines and chlorins. The advantages of this approach are that bacteria can be eradicated in very short periods of time (seconds or minutes), resistance development in the target bacteria is unlikely and damage to adjacent host tissues and disruption of the normal microflora can be avoided. This approach may be a useful alternative to antibiotics and antiseptics in eliminating cariogenic and periodontopathogenic bacteria from disease lesions and for the disinfection of root canals. Not only would this be of benefit for the treatment of these diseases but, by replacing the antimicrobial agents that are currently used for such purposes, it would help to conserve our dwindling supply of antimicrobial agents that are effective in the treatment of serious systemic infections.     

A colorimetric assay for rapid screening of antimicrobial peptides

The increased resistance of various bacteria toward available antibiotic drugs has initiated intensive research efforts into identifying new sources of antimicrobial substances. Short antibiotic peptides (10-30 residues) are prevalent in nature as part of the intrinsic defense mechanisms of most organisms and have been proposed as a blueprint for the design of novel antimicrobial agents. Antimicrobial peptides are generally believed to kill bacteria through membrane permeabilization and extensive pore-formation. Assays providing rapid and easy evaluation of interactions between antimicrobial membrane peptides and lipid bilayers could significantly improve screening for substances with effective antibacterial properties, as well as contribute to the elucidation of structural and functional properties of antimicrobial peptides. Here we describe a colorimetric sensor in which particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids were shown to exhibit striking color changes upon interactions with antimicrobial membrane peptides. The color changes in the system occur because of the structural perturbation of the lipids following their interactions with antimicrobial peptides. The assay was also sensitive to the antibacterial properties of structurally and functionally related peptide analogs.     

Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at ‘a’ and/or ‘d’ position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its ‘a’ and ‘d’ positions with d-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its d-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at ‘a’ and/or ‘d’ positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.     

铁载体-抗生素耦合物:一种新型的抗菌制剂

随着细菌对抗生素耐药性的增强,寻找一种新型抗菌制剂越来越重要。细菌细胞外膜对药物分子的通透性降低是引起致病菌产生耐药性的一个重要因素,克服膜介导耐药性的方法之一是利用铁载体-抗生素耦合物。铁载体是细菌分泌的一种小分子铁离子螯合物,与铁离子螯合后被特定的外膜受体识别并转运至胞浆内供细菌利用。人工合成的铁载体-抗生素耦合物被特定外膜受体识别后主动转运跨过外膜进入胞质内。当铁载体-抗生素耦合物到达细胞质,它们通过释放药物杀死微生物,这可以阻止进一步获取铁离子,并且耦合物自身也可以作为一种抗菌剂。本文综述了铁载体-抗生素耦合物作为一种新型抗菌制剂的研究进展,有助于为进一步研发新型抗菌药物提供理论基础,对治疗耐药性细菌性疾病具有潜在的重要意义。     

Solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells. QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO(2), but also ZnO and others including CuO. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative; in others it is the opposite. These studies have been well reviewed. In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents. CdTe QDs have anti-microbial effects alone or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water. In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent. The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.     

Antimicrobial susceptibility of Edwardsiella ictaluri

Edwardsiella ictaluri isolates collected over the past 7 yr from outbreaks of disease in fish occurring in different geographic areas were screened against 37 antimicrobial agents. Edwardsiella ictaluri were found to be susceptible to most agents active against gram negative bacteria such as aminoglycosides, cephalosporins, the "newer" penicillins, quinolones, tetracyclines, chloramphenicol, nitrofurantoin, and potentiated sulfonamides. Resistance was observed against colistin, sulfonamides, and several agents regarded as effective for gram positive bacteria. There was no evidence of unusual antimicrobial resistance associated with E. ictaluri or of developing resistance.