Bacterial type VII secretion: An important player in host-microbe and microbe-microbe interactions.

Type VII secretion systems (T7SSs) are poorly understood protein export apparatuses found in mycobacteria and many species of Gram-positive bacteria. To date, this pathway has predominantly been studied in Mycobacterium tuberculosis, where it has been shown to play an essential role in virulence; however, much less studied is an evolutionarily divergent subfamily of T7SSs referred to as the T7SSb. The T7SSb is found in the major Gram-positive phylum Firmicutes where it was recently shown to target both eukaryotic and prokaryotic cells, suggesting a dual role for this pathway in host-microbe and microbe-microbe interactions. In this review, we compare the current understanding of the molecular architectures and substrate repertoires of the well-studied mycobacterial T7SSa systems to that of recently characterized T7SSb pathways and highlight how these differences may explain the observed biological functions of this understudied protein export machine.

Efficient Photodynamic Killing of Gram-Positive Bacteria by Synthetic Curcuminoids.

In our previous study, we have demonstrated that curcumin can efficiently kill the anaerobic bacterium Propionibacterium acnes by irradiation with low-dose blue light. The curcuminoids present in natural plant turmeric mainly include curcumin, demethoxycurcumin, and bisdemethoxycurcumin. However, only curcumin is commercially available. Eighteen different curcumin analogs, including demethoxycurcumin and bisdemethoxycurcumin, were synthesized in this study. Their antibacterial activity against Gram-positive aerobic bacteria Staphylococcus aureus and Staphylococcus epidermidis was investigated using the photodynamic inactivation method. Among the three compounds in turmeric, curcumin activity is the weakest, and bisdemethoxycurcumin possesses the strongest activity. However, two synthetic compounds, (1E,6E)-1,7-bis(5-methylthiophen-2-yl)hepta-1,6-diene-3,5-dione and (1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione, possess the best antibacterial activity among all compounds examined in this study. Their chemical stability is also better than that of bisdemethoxycurcumin, and thus has potential for future clinical applications.

Identification of a Novel Cathelicidin from the Deinagkistrodon acutus Genome with Antibacterial Activity by Multiple Mechanisms.

The abuse of antibiotics and the consequent increase of drug-resistant bacteria constitute a serious threat to human health, and new antibiotics are urgently needed. Research shows that antimicrobial peptides produced by natural organisms are potential substitutes for antibiotics. Based on Deinagkistrodonacutus (known as five-pacer viper) genome bioinformatics analysis, we discovered a new cathelicidin antibacterial peptide which was called FP-CATH. Circular dichromatic analysis showed a typical helical structure. FP-CATH showed broad-spectrum antibacterial activity. It has antibacterial activity to Gram-negative bacteria and Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The results of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that FP-CATH could cause the change of bacterial cell integrity, having a destructive effect on Gram-negative bacteria and inducing Gram-positive bacterial surface formation of vesicular structure. FP-CATH could bind to LPS and showed strong binding ability to bacterial DNA. In vivo, FP-CATH can improve the survival rate of nematodes in bacterial invasion experiments, and has a certain protective effect on nematodes. To sum up, FP-CATH is likely to play a role in multiple mechanisms of antibacterial action by impacting bacterial cell integrity and binding to bacterial biomolecules. It is hoped that the study of FP-CATH antibacterial mechanisms will prove useful for development of novel antibiotics.

One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide.

How, when and why the transition between cell envelopes with one membrane (Gram-positives or monoderms) and two (Gram-negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram-positive/Gram-negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of the diderm/monoderm transition.

The Gram-Positive Bacterial Cell Wall.

The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.

A significantly enhanced antibacterial spectrum of D-enantiomeric lipopeptide bactenecin.

Cationic antimicrobial peptides (CAMPs) are important antibiotics because they possess a broad spectrum of activity against both Gram-positive and Gram-negative bacteria, including those resistant to traditional antibiotics. The cyclic peptide bactenecin is a 12-amino acid CAMP that contains one intramolecular disulfide bond. To improve the antibacterial activity of bactenecin, we designed and synthesized several bactenecin analogs by applying multiple approaches, including amino acid substitution, use of the d-enantiomeric form, and lipidation. Among the synthetic analogs, d-enantiomeric bactenecin conjugated to capric acid, which we named dBacK-(cap), exhibited a significantly enhanced antibacterial spectrum with MIC values ranging from 1 to 8 µM against both Gram-positive and Gram-negative bacteria, including some drug-resistant bacteria. Upon exposure to dBacK-(cap), S. aureus cells were killed within 1 h at the MIC value, but full inactivation of E. coli required over 2 h. These results indicate that covalent addition of a d-amino acid and a fatty acid to bactenecin is the most effective approach for enhancing its antibacterial activity.

The effect of chlorination degree and substitution pattern on the interactions of polychlorinated biphenyls with model bacterial membranes.

Polychlorinated biphenyls (PCB) are persistent organic pollutants that due to their chemical resistivity and inflammability found multiple applications. In spite of the global ban for PCB production, due to their long half-lives periods, PCB accumulate in the soils, so effective bioremediation of the polluted lands is of crucial importance. Some of the 209 PCB congeners exhibit increased toxicity to soil bacteria and their presence impoverish the soil decomposer community and slows down the degradation of environmental pollutants in the soils. The exact mechanism of PCB antimicrobial activity is unknown, but it is strictly related with the membrane activity of PCB. Therefore, to shed light on these interactions we applied Langmuir monolayers formed by selected phospholipids as model bacterial membranes. In our studies we tested 5 PCB congeners differing in the degree of chlorination and the distribution of the chlorine substituents around the biphenyl frame. Special attention was paid to tetra-substituted PCB because of their increased presence in the environment and disubstituted PCB being their degradation products. To characterize the model membranes as Langmuir monolayers, we used surface pressure measurements, Brewster angle microscopy and Grazing Incidence X-ray Diffraction. It turned out that among the tetra-substituted PCB the ortho-substituted non-dioxin like compound was much more membrane destructive than the flat dioxin-like congener. On the contrary, among the di-substituted PCB the flat para-substituted 2,2'-dichlorobiphenyl turned out to exhibit high membrane activity.

The antibacterial activity of rhamnolipid biosurfactant is pH dependent.

Rhamnolipid (RL) biosurfactants have been studied as agents to control the growth of food pathogens however, to be successful applied as antimicrobial agent in food, is important to determine the effect of environmental conditions on RL activity. Once pH is a determinant factor to the development of microorganisms in food, we investigated the antimicrobial activity of RL under different pH values. The antimicrobial activity of RL against the Gram-positive pathogens L. monocytogenes, B. cereus and S. aureus was pH dependent and favored at more acidic conditions while the Gram-negative Salmonella enterica and E. coli (EHEC) showed resistance at all pH levels studied. Bacillus cereus was the most sensitive bacteria showing MIC of 19.5 µg/mL and eradication of the population was observed after 30 min with 39.1 µg/mL of RL. The sensitivity to RL was associated with reduction on cell surface hydrophobicity and cytoplasmic membrane damage. Scanning Electron Microscopy images evidenced the cell damage promoted by RL on sensitive strains. The pH is an important factor to be considered on developing RL-based strategies to the control of food pathogens.

Aurone derivatives as promising antibacterial agents against resistant Gram-positive pathogens.

A set of variously substituted aurones was synthesized and evaluated against Methicillin-Resistant S. aureus (MRSA) and P. aeruginosa. Several analogues were found active against MRSA, but no effect was recorded against P. aeruginosa. Compounds 27, 30 and 33 showed low cytotoxicity, and were tested against a full range of bacterial (Gram-positive and Gram-negative) and fungal species, including resistant strains. These aurones displayed a selective inhibition of Gram-positive bacteria with excellent Therapeutic Index values, while showing no significant action on several Gram-negative strains, H. pylori and V. alginolyticus being the only susceptible strains among the Gram-negative bacteria tested. A permeabilization assay showed that the antibacterial activity of at least some of the aurones could be linked to alterations of the bacterial membrane. Overall, this study endorses the use of the aurone scaffold for the development of new potent and selective antibacterial agents.

Similarities and Differences between Silver Ions and Silver in Nanoforms as Antibacterial Agents.

Silver is considered as antibacterial agent with well-known mode of action and bacterial resistance against it is well described. The development of nanotechnology provided different methods for the modification of the chemical and physical structure of silver, which may increase its antibacterial potential. The physico-chemical properties of silver nanoparticles and their interaction with living cells differs substantially from those of silver ions. Moreover, the variety of the forms and characteristics of various silver nanoparticles are also responsible for differences in their antibacterial mode of action and probably bacterial mechanism of resistance. The paper discusses in details the aforementioned aspects of silver activity.

Thymoquinone inhibits biofilm formation and has selective antibacterial activity due to ROS generation.

The present study was aimed to investigate the antibacterial potential and antibiofilm activity of thymoquinone and its mechanism of action. Antibacterial activity of thymoquinone was studied using minimum inhibitory concentration, minimum bactericidal concentration, time-kill assay, and post-antibiotic effect. Thymoquinone exhibited antibacterial activity against both Gram-negative and Gram-positive bacteria. In this study, the minimum inhibitory concentration was found to be in the range of 1.56 to 100 µg/ml. Scanning electron microscopy imaging revealed changes in cell morphology with dents, cell lysis, and reduction in cell size. Live/dead imaging using acridine orange and ethidium bromide confirmed the bactericidal activity as treated bacteria showed selective uptake of ethidium bromide over acridine orange. Cell viability was also studied using HaCaT (human keratinocytes) cell line by MTT assay, and IC90 value was found to be 50 µg/ml. This IC90 value was higher than that of MICbacteria (except for MIC of E. coli), demonstrating that its selectivity is higher towards bacteria than normal human cells. Thymoquinone also showed promising antibiofilm activity against Gram-negative (E. coli and P. aeruginosa) and Gram-positive bacteria (B. subtilis and S. aureus), which was studied by crystal violet assay, CFU method, and SEM. For understanding the mechanism of action of thymoquinone, DiSC3, NPN, and ROS assay was performed. DiSC3 and NPN assay has not shown any membrane damage whereas bacterial cells treated with thymoquinone at MIC showed increased dichlorofluorescin fluorescence, suggesting that the probable mechanism of action of thymoquinone against bacterial cells is due to the production of reactive oxygen species.

Biosynthesized silver nanoparticles: are they effective antimicrobials?

BACKGROUND: Silver nanoparticles (AgNPs) are increasingly being used in medical applications. Therefore, cost effective and green methods for generating AgNPs are required. OBJECTIVES: This study aimed towards the biosynthesis, characterisation, and determination of antimicrobial activity of AgNPs produced using Pseudomonas aeruginosa ATCC 27853. METHODS: Culture conditions (AgNO3 concentration, pH, and incubation temperature and time) were optimized to achieve maximum AgNP production. The characterisation of AgNPs and their stability were evaluated by UV-visible spectrophotometry and scanning electron microscopy. FINDINGS: The characteristic UV-visible absorbance peak was observed in the 420-430 nm range. Most of the particles were spherical in shape within a size range of 33-300 nm. The biosynthesized AgNPs exhibited higher stability than that exhibited by chemically synthesized AgNPs in the presence of electrolytes. The biosynthesized AgNPs exhibited antimicrobial activity against Escherichia coli, P. aeruginosa, Salmonella typhimurium, Staphylococcus aureus, methicillin-resistant S. aureus, Acinetobacter baumannii, and Candida albicans. MAIN CONCLUSION: As compared to the tested Gram-negative bacteria, Gram-positive bacteria required higher contact time to achieve 100% reduction of colony forming units when treated with biosynthesized AgNPs produced using P. aeruginosa.

Protease Hydrolysates of Filefish (Thamnaconus modestus) Byproducts Effectively Inhibit Foodborne Pathogens.

In this study, novel antimicrobial peptides had been derived by enzymatic hydrolysis of filefish (Thamnaconus modestus) byproduct (HFBP). Different proteases, (papain [P], trypsin [T], neutrase [N], pepsin [PE], and the mixture I [PN] and mixture II [PT]) and different hydrolysis time (60, 120, 180, and 240 min), have been used to generate peptides with different lengths and amino acid sequences. The antimicrobial properties of HFBPs were tested, against Gram-positive and Gram-negative strains, using disc diffusion method. HFBP prepared after 120 min of the enzymatic hydrolysis by trypsin (HFBP-T) exhibited greatest antibacterial activities. Bacillus cereus 10451 (BC) and Salmonella enteritidis 10982 (SE) strains were most sensitive to HFBP-T with an inhibitory zone of 24.68 and 29.67 mm diameter and minimum inhibitory concentration of 1.25 and 2.5 mg/mL, respectively. Moreover, the antimicrobial activities of tested HFBPs increased significantly at low pH and temperature. The amino acid analysis showed that HFBP-T protein hydrolysate is high in an amino acid of proline, which probably contributes to the antimicrobial activity. The results obtained from scanning electron microscopy suggested that HFBPs might kill bacteria by acting on the cell wall of bacteria. Conclusively, the HFBP derived from filefish byproduct with biological activates is an interesting alternative to the use of waste from the fishing industry as natural antimicrobials in food stuff.

Bactericidal Effect of Pterostilbene Alone and in Combination with Gentamicin against Human Pathogenic Bacteria.

The antibacterial activity of pterostilbene in combination with gentamicin against six strains of Gram-positive and Gram-negative bacteria were investigated. The minimum inhibitory concentration and minimum bactericidal concentration of pterostilbene were determined using microdilution technique whereas the synergistic antibacterial activities of pterostilbene in combination with gentamicin were assessed using checkerboard assay and time-kill kinetic study. Results of the present study showed that the combination effects of pterostilbene with gentamicin were synergistic (FIC index < 0.5) against three susceptible bacteria strains: Staphylococcus aureus ATCC 25923, Escherichia coli O157 and Pseudomonas aeruginosa 15442. However, the time-kill study showed that the interaction was indifference which did not significantly differ from the gentamicin treatment. Furthermore, time-kill study showed that the growth of the tested bacteria was completely attenuated with 2 to 8 h treatment with 0.5 × MIC of pterostilbene and gentamicin. The identified combinations could be of effective therapeutic value against bacterial infections. These findings have potential implications in delaying the development of bacterial resistance as the antibacterial effect was achieved with the lower concentrations of antibacterial agents.

Antibacterial compound produced by Pseudomonas aeruginosa strain UICC B-40, an endophytic bacterium isolated from Neesia altissima.

This study's aim was to determine the identity of antibacterial compounds produced by Pseudomonas aeruginosa strain UICC B-40 and describe the antibacterial compounds' mechanisms of action for damaging pathogenic bacteria cells. Isolation and identification of the compounds were carried out using thin layer chromatography (TLC), nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LC-MS) analyses. Antibacterial activity was assayed via minimum inhibitory concentration (MIC) and the antibacterial compound mechanism was observed morphologically through scanning electron microscopy (SEM). This study successfully identified the (2E,5E)-phenyltetradeca-2,5-dienoate antibacterial compound (molecular weight 300 g/mol), composed of a phenolic ester, fatty acid and long chain of aliphatic group structures. MIC values for this compound were determined at 62.5 µg/ml against Staphylococcus aureus strain ATCC 25923. The mechanism of the compound involved breaking down the bacterial cell walls through the lysis process. The (2E,5E)-phenyltetradeca-2,5-dienoate compound exhibited inhibitory activity on the growth of Gram-positive bacteria.

Antibiofilm efficacy of green synthesized graphene oxide-silver nanocomposite using Lagerstroemia speciosa floral extract: A comparative study on inhibition of gram-positive and gram-negative biofilms.

Biofilm architecture provides bacteria with enhanced antibiotic resistance, thus raising the need to search for alternative therapies that can inhibit the bacterial colonization. In the present study, we synthesized graphene oxide-silver nanocomposite (GO-Ag) by non-toxic and eco-friendly route using a floral extract of Legistromia speciosa (L.) Pers. The gas chromatography-mass spectrometry (GC-MS) analysis of plant extract revealed the presence of compounds which can simultaneously act as reducing and capping agents. The sub-inhibitory concentrations of synthesized GO-Ag reduced the biofilm formation in both gram-negative (E. cloacae) and gram-positive (S. mutans) bacterial models. Growth curve assay, membrane integrity assay, scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) revealed different mechanisms of biofilm inhibition in E. cloacae and S. mutans. Moreover, quantitative RT-PCR (qRT-PCR) results suggested GO-Ag is acting on S. mutans biofilm formation cascade. Biofilm inhibitory concentrations GO-Ag were also found to be non-toxic against HEK-293 (human embryonic kidney cell line). The whole study highlights the therapeutic potential of GO-Ag to restrain the onset of biofilm formation in bacteria.

Envelope Structures of Gram-Positive Bacteria.

Gram-positive organisms, including the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, have dynamic cell envelopes that mediate interactions with the environment and serve as the first line of defense against toxic molecules. Major components of the cell envelope include peptidoglycan (PG), which is a well-established target for antibiotics, teichoic acids (TAs), capsular polysaccharides (CPS), surface proteins, and phospholipids. These components can undergo modification to promote pathogenesis, decrease susceptibility to antibiotics and host immune defenses, and enhance survival in hostile environments. This chapter will cover the structure, biosynthesis, and important functions of major cell envelope components in gram-positive bacteria. Possible targets for new antimicrobials will be noted.

Bacillus methylotrophicus Strain NKG-1, Isolated from Changbai Mountain, China, Has Potential Applications as a Biofertilizer or Biocontrol Agent.

Chemical pesticides are widely used in agriculture, which endangers both environmental health and food safety. Biocontrol is an environmentally-friendly and cost-effective green technique in environmental protection and agricultural production; it generally uses selected bioresources, including beneficial microorganisms. We isolated a novel bacterial strain (NKG-1) from the rare dormant volcanic soils of Changbai Mountain in China's Jilin Province. The strain was identified as Bacillus methylotrophicus using morphological, biochemical, physiological, and phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi on solid media. A greenhouse experiment showed that application of NKG-1 fermentation broth prior to inoculation of Botrytis cinerea, the cause of gray tomato mold, inhibited growth of the mold by 60%. Furthermore, application of a 100× dilution of NKG-1 fermentation broth to tomato seedlings yielded a significant increase in seedling fresh weight (27.4%), seedling length (12.5%), and root length (57.7%) compared to the control. When the same dosage was applied in the field, we observed increases in tomato plant height (14.7%), stem diameter (12.7%), crown width (16.3%), and maximum fruit diameter (11.5%). These results suggest that NKG-1 has potential commercial application as a biofertilizer or biocontrol agent.

New insights into the mode of action of the lantibiotic salivaricin B.

Salivaricin B is a 25 amino acid polycyclic peptide belonging to the type AII lantibiotics and first shown to be produced by Streptococcus salivarius. In this study we describe the bactericidal mode of action of salivaricin B against susceptible Gram-positive bacteria. The killing action of salivaricin B required micro-molar concentrations of lantibiotic whereas the prototype lantibiotic nisin A was shown to be potent at nano-molar levels. Unlike nisin A, salivaricin B did not induce pore formation or dissipate the membrane potential in susceptible cells. This was established by measuring the fluorescence of the tryptophan residue at position 17 when salivaricin B interacted with bacterial membrane vesicles. The absence of a fluorescence blue shift indicates a failure of salivaricin B to penetrate the membranes. On the other hand, salivaricin B interfered with cell wall biosynthesis, as shown by the accumulation of the final soluble cell wall precursor UDP-MurNAc-pentapeptide which is the backbone of the bacterial peptidoglycan. Transmission electron microscopy of salivaricin B-treated cells showed a reduction in cell wall thickness together with signs of aberrant septum formation in the absence of visible changes to cytoplasmic membrane integrity.

Long-term antibiofilm activity of carboxymethyl chitosan on mixed biofilm on silicone.

OBJECTIVES/HYPOTHESIS: Silicone voice prostheses are most frequently used in voice rehabilitation of laryngectomized patients. However, the functional device lifetimes are limited due to formation of mixed biofilms. Existing in vitro models simulating biofilm formation are restricted to only short-term periods. STUDY DESIGN: The goal of this study was to determine the effect of carboxymethyl chitosan on mixed biofilm formation of fungi and bacteria on silicone over a long-term period. METHODS: Mixed species biofilms of Candida albicans, Candida tropicalis, Lactobacillus gasseri, Streptococcus salivarius, Rothia dentocariosa, and Staphylococcus epidermidis were cultivated on the surfaces of medical-grade silicone with and without addition of carboxymethyl chitosan. Biofilm kinetics was monitored using specially designed image analysis software to calculate the percentual surface covering of each platelet. Biofilm architecture was investigated by scanning electron microscopy. RESULTS: A cover of living mixed biofilm could be generated over 22 days on silicone and the maximum of 22% biofilm surface covering at day 22. However, less than 4% surface coverage was observed on the carboxymethyl chitosan-treated plates in the testing period. Scanning electron microscopy confirms that, on surfaces treated by carboxymethyl chitosan, the biofilm was less dense. In addition, there were fewer layers of cells and profuse cellular debris, together with degrading and morphologically altered yeast cells. CONCLUSION: Carboxymethyl chitosan may serve as a possible antibiofilm agent to limit biofilm formation on voice prostheses. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E404-E408, 2016.