Gram-negative Bacterial Species Research Articles

Characterization of Endophytic Streptomyces griseorubens MPT42 and Assessment of Antimicrobial Synergistic Interactions of its Extract and Essential Oil from Host Plant Litsea cubeba.

The present study aimed to evaluate the synergistic effects of the crude ethyl acetate extract (CEAE) from endophytic actinomycete MPT42 and essential oil (EO) of the same host plant Litsea cubeba. The isolate MPT42, exhibiting broad-spectrum antimicrobial activities and harboring all three antibiotic-related biosynthetic genes pks-I, pks-II, and nrps, was identified as Streptomycete griseorubens based on an analysis of the morphology, physiology, and 16S rDNA sequence. Minimum inhibitory concentrations (MICs) and the fractional inhibitory concentration index were used to estimate the synergistic effects of various combined ratios between CEAE or antibiotics (erythromycin, vancomycin) and EO toward 13 microbial strains including pathogens. L. cubeba fruit EO, showing the main chemical constituents of 36.0% citral, 29.6% carveol, and 20.5% limonene, revealed an active-low against tested microbes (MICs ≥ 600 μg/mL). The CEAE of S. griseorubens culture exhibited moderate–strong antimicrobial activities against microbes (MICs = 80–600 μg/mL). Analysis of the mechanism of action of EO on Escherichia coli ATCC 25922 found that bacterial cells were dead after 7 h of the EO treatment at 1 MIC (5.5 mg/mL), where 62% cells were permeabilized after 2 h and 3% of them were filament (length ≥ 6 μm). Combinations of CEAE, erythromycin, or vancomycin with EO led to significant synergistic antimicrobial effects against microbes with 4–16 fold reduction in MIC values when compared to their single use. Interestingly, the vancomycin–EO combinations exhibited a strong synergistic effect against five Gram-negative bacterial species. This could assume that the synergy was possibly due to increasing the cell membrane permeability by the EO acting on the bacterial cells, which allows the uptake and diffusion of antimicrobial substances inside the cell easily. These findings in the present study therefore propose a possible alternative to combat the emergence of multidrug-resistant microbes in veterinary and clinics.

How Oxygen Availability Affects the Antimicrobial Efficacy of Host Defense Peptides: Lessons Learned from Studying the Copper-Binding Peptides Piscidins 1 and 3.

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we performed growth inhibition and time-kill assays against C. difficile showing that both piscidins suppress proliferation of C. difficile by killing bacterial cells. Microscopy experiments show that the peptides accumulate at sites of membrane curvature. We find that both piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. Notably, copper does not enhance piscidin activity against C. difficile. Thus, while antimicrobial activity of piscidin peptides is conserved in aerobic and anaerobic settings, the peptide–copper interaction depends on environmental oxygen to achieve its maximum potency. The development of pharmaceuticals from HDPs such as piscidin will necessitate consideration of oxygen levels in the targeted tissue.

Bacterial Killing Activity of Polymorphonuclear Myeloid-Derived Suppressor Cells Isolated From Tumor-Bearing Dogs.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are implicated in the progression and outcome of a variety of pathological states, from cancer to infection. Our previous work has identified three antimicrobial peptides differentially expressed by PMN-MDSCs compared to conventional neutrophils isolated from dogs, mice, and human patients with cancer. We therefore hypothesized that PMN-MDSCs in dogs with cancer possess antimicrobial activity. In the current work, we observed that exposure of PMN-MDSCs to Gram-negative bacteria (Escherichia coli) increased the expression of reactive oxygen species by the PMN-MDSCs, indicating that they are capable of initiating an anti-microbial response. Electron microscopy revealed that the PMN-MDSCs phagocytosed Gram-negative and Gram-positive (Staphylococcus aureus) bacterial species. Lysis of bacteria within some of the PMN-MDSCs suggested bactericidal activity, which was confirmed by the recovery of significantly lower numbers of bacteria of both species following exposure to PMN-MDSCs isolated from tumor-bearing dogs. Our data therefore indicate that PMN-MDSCs isolated from dogs with cancer, in common with PMNs, have phagocytic and bactericidal activity. This nexus of immunosuppressive and antimicrobial activity reveals a hitherto unrecognized function of MDSCs.

Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers

Infection of various pathogenic bacteria causes severe illness to human beings. Despite the research advances, current identification tools still exhibit limitations in detecting Gram-negative bacteria with high accuracy. In this study, we isolated single-stranded DNA aptamers against multiple Gram-negative bacterial species using Toggle-cell-SELEX (systemic evolution of ligands by exponential enrichment) and constructed an aptamer-based detection tool towards bacterial secretory cargo released from outer membranes of Gram-negative bacteria. Three Gram-negative bacteria, Escherichia coli DH5α, E. coli K12, and Serratia marcescens, were sequentially incubated with the pool of random DNA sequences at each SELEX loop. Two aptamers selected, GN6 and GN12, were 4.2-times and 3.6-times higher binding to 108 cells of Gram-negative bacteria than to Gram-positive bacteria tested, respectively. Using GN6 aptamer, we constructed an Enzyme-linked aptamer assay (ELAA) to detect bacterial outer membrane vesicles (OMVs) of Gram-negative bacteria, which contain several outer membrane proteins with potent immunostimulatory effects. The GN6-ELAA showed high sensitivity to detect as low as 25 ng/mL bacterial OMVs. Aptamers developed in this study show a great potential to facilitate medical diagnosis and early detection of bacterial terrorism, based on the ability to detect bacterial OMVs of multiple Gram-negative bacteria.

Pyridine azo disperse dye derivatives and their selenium nanoparticles (SeNPs): synthesis, fastness properties, and antimicrobial evaluations.

AimAiming to produce pyridine azo disperse dyes with good fastness properties and promising antimicrobial activity, a number of novel systems of polyfunctionalized pyridine azo dyes and their selenium nanoparticles (SeNPs) were synthesized.Materials and methodsThe synthesized products were formed by the reaction of diazotized aniline derivatives or diazotized amino antipyrene with any of dibenzoyl methane or benzoyl acetone and cyanoacetamide in boiling ethanolic sodium ethoxide. The structures of the newly synthesized compounds were elucidated by elemental analysis and spectral data. Moreover, (SeNPs) of the pyridine azo disperse dyes were characterized by Ultra-Violet -Visible spectrophotometry, dynamic light scattering , X-ray diffraction, and transmission electron microscope analysis. On the other hand, the synthesized dyes and its (SeNPs) were applied for disperse dyeing of nylon 66 and their fastness properties were measured, such as washing, rubbing, perspiration, and light fastness. In addition, the antimicrobial activities for all the synthesized compounds and for (SeNPs) prepared compounds (2bN, 2cN, 2fN, 2gN, 2hN) were evaluated.ResultsCompounds 2bN, 2c, 2cN, 2fN, 2gN, 2h, 2hN, and 2i were the most active compounds against all Gram-positive and Gram-negative bacterial species. While, compounds 2b, 2f, 2g, and 5b were the most active toward some of the bacterial strains (at least two from the selected four strains). Moreover, compounds 2bN, 2cN, 2fN, 2gN, 2h, 2hN showed higher activity toward the fungal strain. Also, the minimal inhibitory concentrations for all the most active compounds were determined.ConclusionFinally, all the (SeNPs) compounds revealed higher activity against bacterial and fungal strains than the other synthesized compounds.

Antioxidant, antimicrobial and allelopathic activities and surface disinfection of the extract of Psidium cattleianum sabine leaves

The objective this study was to obtain extracts, using ultrasound and enzymes, of leaves of Psidium. cattleianum Sabine, collected in the summer and winter, and evaluate the antioxidant, allelopathic and antimicrobial activities. Photomicrographs of leaf structure were obtained by SEM. The phenolic compounds content (TPC) and the antioxidant activity were determined. The allelopathic activity was verified in bioassays for each of the extracts against the test plant (Lactuca sativa). The antimicrobial activity was determined against Gram-positive and Gram-negative bacterial species. It was evaluated the use of extracts as disinfectants. The SEM showed that the extraction form affected the cell structure of the leaves but did not influence the TPC. The highest TPC was found in leaf extracts collected during winter, with values between 123 and 144 mg EAG.g−1. The level of antioxidant activity estimated presented higher value for the leaves collected in the summer, where the IC50 presented values of 37 μg mL−1. The results also indicate that all extracts tested show considerable allelopathic activity, with inhibition of growth of more than 50%. The antimicrobial activity can be classified as partially active to very active, where the best results were found against L. monocytogenes, with inhibition halos measuring up to 23 mm. It was also verified that the time of collection of leaves interfered in the values found. With regard to surface disinfection, all the extracts tested positive results. Experiments have shown that extracts of P. cattleianum Sabine are a source of bioactive compounds, which have antioxidant, allelopathic and antibacterial activity.

The ubiquitin-like modification by ThiS and ThiF in Escherichia coli

Escherichia coli, one of the most well-studied gram-negative bacterial species, encodes two ubiquitin-like proteins (UBLs), ThiS and MoaD. The studies on prokaryotic UBLs such as Pup, and small archaeal modifier protein have revealed the function of UBLs. However, in gram-negative bacteria, the functions of UBLs in protein modification are still poorly understood to date. Here, we report that ThiS, which has a β-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, is able to form protein conjugates in vivo and in vitro. We also constructed in vitro ThiS conjugation (thisylation) system and identified the modified lysine sites by MS/MS, this provides an essential platform for studying the UBLs thisylation system in E. coli. The modification system is dependent on lysine 83 (ATPase activity site) and cysteine 169 (zinc binding site) in ThiF and three important substrates, GroEL, PriC, FtsA, were found to be covalently modified by this system in vitro. Taken together, this study provided evidence that the protein conjugation function of β-grasp fold UBLs is conserved in the three major evolutionary lineages of life.

Antimicrobial and Immune-Modulatory Effects of Vitamin D Provide Promising Antibiotics-Independent Approaches to Tackle Bacterial Infections - Lessons Learnt from a Literature Survey.

Antimicrobial multidrug-resistance (MDR) constitutes an emerging threat to global health and makes the effective prevention and treatment of many, particularly severe infections challenging, if not impossible. Many antibiotic classes have lost antimicrobial efficacy against a plethora of infectious agents including bacterial species due to microbial acquisition of distinct resistance genes. Hence, the development of novel anti-infectious intervention strategies including antibiotic-independent approaches is urgently needed. Vitamins such as vitamin D and vitamin D derivates might be such promising molecular candidates to combat infections caused by bacteria including MDR strains. Using the Pubmed database, we therefore performed an in-depth literature survey, searching for publications on the antimicrobial effect of vitamin D directed against bacteria including MDR strains. In vitro and clinical studies between 2009 and 2019 revealed that vitamin D does, in fact, possess antimicrobial properties against both Gram-positive and Gram-negative bacterial species, whereas conflicting results could be obtained from in vivo studies. Taken together, the potential anti-infectious effects for the antibiotic-independent application of vitamin D and/or an adjunct therapy in combination with antibiotic compounds directed against infectious diseases such as tuberculosis, H. pylori infections, or skin diseases, for instance, should be considered and further investigated in more detail.

Synthesis, Characterization, and Theoretical Studies of the New Antibacterial Zn(II) Complexes from New Fluorescent Schiff Bases Prepared by imidazo[4',5':3,4]benzo[1,2-c]isoxazole

The novel fluorescentheterocyclic bidentate ligands have been synthesized by the high yields reaction of 8-(4-chlorophenyl)-3-Iso-butyl-3H-imidazo[4',5':3,4]benzo[1,2-c]isoxazol-5-amine with p-hydroxybenzaldehyde and p-chlorobenzaldehyde. The ligands reacted with Zn(II) ion to gained novel complexes. The optical properties of these structures were checked and the outcomes represented that they showed interesting photophysical properties. Optimized geometries and assignment of the IR bands and NMR chemical shifts of the new complexes were also computed by using Density Functional Theory (DFT) methods that were in good agreement with the experimental values, confirming the suitability of the optimized geometries for Zn(II) complexes. These new compounds have shown potent antibacterial properties and their antibacterial activity (MIC) against Gram-positive and Gram-negative bacterial species were also specific.

Biosynthesis and Bioactivity of Prodiginine Analogs in Marine Bacteria, Pseudoalteromonas: A Mini Review.

The Prodiginine family consists of primarily red-pigmented tripyrrole secondary metabolites that were first characterized in the Gram-negative bacterial species Serratia marcescens and demonstrates a wide array of biological activities and applications. Derivatives of prodiginine have since been characterized in the marine γ-proteobacterium, Pseudoalteromonas. Although biosynthetic gene clusters involved in prodiginine synthesis display homology among genera, there is an evident structural difference in the resulting metabolites. This review will summarize prodiginine biosynthesis, bioactivity, and gene regulation in Pseudoalteromonas in comparison to the previously characterized species of Serratia, discuss the ecological contributions of Pseudoalteromonas in the marine microbiome and their eukaryotic hosts, and consider the importance of modern functional genomics and classic DNA manipulation to understand the overall prodiginine biosynthesis pathway.

Lateral flow biosensor combined with loop-mediated isothermal amplification for simple, rapid, sensitive, and reliable detection of Brucella spp.

Brucella species is responsible for brucellosis in human and animals, which is still of public health, veterinarian, and economic concern in many regions of the world. Here, a novel molecular diagnosis assay, termed loop-mediated isothermal amplification coupled with nanoparticles-based lateral flow biosensor (LAMP-LFB), was developed and validated for simply, rapidly, and reliably detecting all Brucella spp. strains. A set of six primers was designed based on the Brucella-specific gene Bscp31. The Brucella-LAMP results were visually reported by biosensor within 2 mins. A variety of bacterial strains representing several Brucella species, as well as several Gram-negative and Gram-positive bacterial species were used to determine the analytical sensitivity and specificity of the assay. Optimal LAMP conditions were 63°C for 40 mins, and the assay’s sensitivity was found to be 100 fg of genomic DNA in the pure cultures. No cross-reactions to non-Brucella strains were obtained; thus, analytical specificity of LAMP-LFB assay is of 100%. Using the protocol, 20 mins for rapid DNA preparation followed by isothermal amplification (40 mins) combined with biosensor detection (2 mins) resulted in a total assay time of approximately 65 mins. In the case of 117 whole blood samples, 13 (11.11%) samples were Brucella-positive by LAMP-LFB, and the diagnostic accuracy was 100% when compared to the culture-biotechnical method. In conclusion, Brucella-LAMP-LFB technique developed in this study is a sensitive and specific method to rapidly identify all Brucella spp. strains, and can be applied as a potential diagnostic tool for brucellosis in basic, clinical, and field laboratories.

Comparison of Carbapenem-Resistant Microbial Pathogens in Combat and Non-combat Wounds of Military and Civilian Patients Seen at a Tertiary Military Hospital, Philippines (2013–2017)

Bacterial wound infections are a danger to both military and civilian populations. The nature of injury and infection associated with combat related wounds are important in guiding antibiotic prophylaxis and empiric treatment guidelines. The isolates were screened for drug-resistance by the MicroScan Walkaway Plus System using either the Negative Breakpoint Combo Panel (NBCP) 30 or 34 or Positive Breakpoint Combo Panel (PBPC) 20 or 23. Isolates with a minimum inhibitory concentration (MIC) of ≥8 μg/mL to imipenem and/or meropenem were tested for both carbapenemase production using the CarbaNP test and real-time PCR to determine molecular resistance mechanisms. Plasmid conjugation analysis was performed to define potential for horizontal gene transfer. We characterized 634 bacterial wound isolates collected from September 2013 to December 2017 from patients seen at a Philippine military tertiary hospital presenting with combat or non-combat injuries [354 (military) and 280 (civilians)]. Staphylococcus aureus was the most predominant bacterial species isolated from wounds in both populations (104/634, 16%). A variety of Gram-negative bacterial species comprised 442/634 (70%) of the isolates identified, with the most prevalent shown to be Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter sp. Carbapenemase production was detected in 34/442 (8%) Gram-negative isolates. Testing for molecular resistance mechanisms showed 32/34 (17 military, 15 civilian) wound isolates were blaNDM positive and 2 were blaVIM positive, with the two blaVIM isolates found in the civilian population. Plasmid conjugation of 14 blaNDM and 2 blaVIM positive wound isolates representatives showed 2/16 (13%) produced E. coli J53 transconjugants (E. coli from a civilian; E. cloacae from a military). We describe in this study the wound bacterial and antibiotic resistance profile in the military (combat vs non-combat associated) and civilian population. We observed that, with the exception of Acinetobacter sp., resistance of prevalent Gram-negative bacterial species to imipenem or meropenem were not significantly different between the military and civilian populations. We also presented data on the prevalent bacterial species isolated from both combat and non-combat wounds in a military tertiary care hospital setting as well as the carbapenemase-encoding gene primarily responsible for carbapenem resistance as well as evidence of horizontal transfer via mobile genetic elements. Clinicians may use this information to guide empiric antibiotic coverage for the predominant organisms if wound culture results are not readily available.A prospective, longitudinal evaluation of the wound bacterial profile documenting the changing bacterial flora using higher resolution molecular strategies can provide a more comprehensive understanding of the diversity, composition, and abundance of bacterial composition of the wound microbial community from the time of injury, during the course of evacuation from the field to higher level of care facilities, and up to wound resolution.

Grevillea juncifolia Hook. and Grevillea robusta A. Cunn. Ex. R. Br. Methanolic Leaf and Flower Extracts Inhibit the Growth of Gram Positive and Gram Negative Bacteria

Introduction: The development of multi-antibiotic resistant strains of bacteria has necessitated the search for new effective antibacterial therapies. Several Grevillea spp. were used traditionally to treat pathogenic illness and are rich in phytocompounds with antibacterial activity. Despite this, the antibacterial activity of Australian Grevillea spp. extracts have not been extensively examined. Methods: The ability of G. juncifolia and G. robusta leaf and flower extracts to inhibit the growth of gram-negative and gram-positive bacterial species and some fungi was investigated by disc diffusion assays. The growth inhibitory activity was further quantified by MIC determination. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: The G. juncifolia and G. robusta extracts were good inhibitors of the growth of both gram-positive and gram-negative bacteria yet were completely ineffective against all fungal species tested. The leaf extracts generally had better antibacterial activity than the flower extracts. The G. juncifolia leaf extract was a particularly good inhibitor of A. faecalis, P. fluorescens, Y. entercolitica and B. subtilis growth, with MIC values of 62, 533, 736 and 682µg/mL respectively. The G. robusta leaf extract was a potent inhibitor of B. cereus and B. subtilis growth (145 and 83 µg/mL respectively). That extract was also a good inhibitor of A. faecalis, P. fluorescens, S. salford, S. aureus and S. epidermidis growth, albeit with substantially higher MIC values. In contrast, none of the extracts inhibited fungal growth. All extracts were determined to be non-toxic in the Artemia franciscana nauplii bioassay, indicating their safety for the treatment of bacterial infections. Conclusion: The lack of toxicity of the G. juncifolia and G. robusta extracts and their growth inhibitory bioactivity against gram-positive and gram-negative bacteria indicate their potential in the development of new antibiotic chemotherapies.

Occurrence of bacteria and endotoxins in fermented foods and beverages from Nigeria and South Africa

In Africa, fermented foods and beverages play significant roles in contributing to food security. Endotoxins are ubiquitous heat stable lipopolysaccharide (LPS) complexes situated in the outer cell membranes of Gram-negative bacteria. This study evaluated the microbiological quality of fermented foods (ogiri, ugba, iru, ogi and ogi baba) and beverages (mahewu and umqombothi) from selected Nigerian and South African markets. The bacterial diversity of the fermented foods was also investigated and the identity of the isolates confirmed by biochemical and molecular methods. Isolate grouping was established through hierarchal clustering and the samples were further investigated for endotoxin production with the chromogenic Limulus Amoebocyte Lysate assay. The total aerobic count of the samples ranged from 5.7 to 10.8 Log CFU/g. Fourteen bacteria genera were detected with most of the isolates being members of the Enterobacteriaceae family. Sphingomonas paucimobilis and Escherichia coli were the dominant Gram-negative bacterial species detected. There were considerable variations in the concentrations of endotoxins produced and the lowest endotoxin concentration was found in ogi (4.3 × 10 EU/g) and the highest in iru (5.5 × 104 EU/g) while, 44% of umqombothi samples had endotoxins. Ogi baba samples had better microbial quality than other samples due to its reduced bacterial load and endotoxin levels. There was a strong positive (r = 0.714, r = 0.996) and significant (p < 0.01) correlation between the endotoxin levels and bacterial loads of the samples. Some previously unreported species of bacteria found in the fermented foods included Aeromonas haemolyticus and Rhizobium radiobacter. This is the first comprehensive report on endotoxins in fermented foods and beverages in Africa. Furthermore, the occurrence of pathogenic bacteria and toxins in the foods and beverages is of serious concern that calls for immediate action.

Generalized and Prolonged Use of Gentamicin-Lock Therapy Reduces Hemodialysis Catheter-Related Infections Due to Gram Negatives

Introduction: The incidence of catheter-related bloodstream infections (CRBSI) ranges from 2.2 to 5.5 episodes per 1,000 catheter-days. Our aim was to evaluate the utility of a generalized and prolonged gentamicin-lock therapy in patients undergoing hemodialysis (HD) in a third-level hospital for the reduction in CRBSI. Methods: A prospective cohort analyzed before and after intervention. During intervention periods after each HD-session, the catheter lumens were locked with gentamicin/heparin for all patients compared to nonintervention periods were the same procedure was performed without gentamicin. Active surveillance was performed for HD CRBSI. Microbiologic assessment and epidemiological data were gathered. Continuous hand hygiene and water quality monitoring were performed. Results: The rates of CRBSI were reduced from 1.28 to 0.2 cases per 1,000 catheter-days when the lock therapy was employed (p = 0.001) The greatest reduction was for CRBSI caused by Pseudomonas aeruginosa were no cases were recorded during the intervention periods (p = 0.001). There was a significant reduction in the total number of isolates; Gram-negative bacterial species (–97.2%) and Gram-positive bacterial species (–61.5%) although only the former reached statistical significance (p = 0.0001). The difference in the absolute risk reduction was 20.56% (95% CI 14.46–26.66%), the calculated Number Needed to Treat was 5 (95% CI 3.8–6.9). No adverse effects were noted. Conclusion: In the current study, gentamicin-lock therapy was associated with a significant reduction in CRBSI specially with P. aeruginosa and other Gram-negative bacteria. It proved to be safe and effective intervention when applied to the entire population of HD patients.

IN VITRO SCREENING OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF MEDICINAL PLANTS GROWING IN SLOVAKIA

Traditional medicine usage at the preventive medicine level is comprehensive and plant-based remedies are being prescribed for the treatment of a large scale of diseases by indigenous medical practitioners around the world. The phytochemicals being observed in the fruits and vegetables as well as herbal plants are getting investigated intensively as their important role in the avoidance of diverse human diseases is undoubtful. Antioxidant and antimicrobial activities of selected medicinal herbs were evaluated in this study. Aerial parts of plants from Hypericaceae, Lamiaceae, Fabaceae, Plantaginaceae, Rubiaceae growing wild in Slovakia (locality Zobor) were investigated to characterize their polyphenol content, flavonoid content, as well as antioxidant and antimicrobial activity. Inspection of the metabolic profile combined with HPLC data showed that the ethanol extract contained mainly eight polyphenols with apigenin, daidzein, kaempferol, rutin, quercetin, vitexin, and cynaroside as the main chemicals. Among them, neochlorogenic acid, chlorogenic acid, trans-p-caffeic acid, trans-p-coumaric acid, trans-p-sinapic acid, trans-p-ferulic acid, rosmarinic acid were identified. The antioxidant capacity of ethanol extracts was evaluated by employing DPPH radical scavenging and phosphomolybdenum assays. All samples were also tested against Gram-positive (Staphylococcus aureus, Bacillus thuringiensis, Micrococcus luteus, Staphyloccocus epidermidis) and Gram-negative (Citrobacter koseri, Escherichia coli, Pseudomonas proteolytica, Hafnia alvei, Salmonella enterica, Yersinia enterocolitica) bacterial species using the disk diffusion and minimum inhibitory concentration assays

Rapid nondestructive measurement of bacterial cultures with 3D interferometric imaging

The agar culture plate has played a crucial role in bacteriology since the origins of the discipline and is a staple bioanalytical method for efforts ranging from research to standard clinical diagnostic tests. However, plating, inoculating, and waiting for microbes to develop colonies that are visible is time-consuming. In this work, we demonstrate white-light interferometry (WLI) as a practical tool for accelerated and improved measurement of bacterial cultures. High resolution WLI surface profile imaging was used for nondestructive characterization and counting of bacterial colonies on agar before they became visible to the naked eye. The three-dimensional (3D) morphology of Gram-negative (Pseudomonas fluorescens) and Gram-positive (Bacillus thuringiensis) bacterial species were monitored with WLI over time by collecting surface profiles of colonies on agar plates with high vertical resolution (3–5 nanometers) and large field of view (3–5 mm). This unique combination of sensitive vertical resolution and large field of view uniquely provided by WLI enables measurement of colony morphologies and nondestructive monitoring of hundreds of microcolonies. Individual bacteria were imaged within the first few hours after plating and colonies were accurately counted with results comparing favorably to counts made by traditional methods that require much longer wait times. Nondestructive imaging was used to track single cells multiplying into small colonies and the volume changes over time in these colonies were used to measure their growth rates. Based on the results herein, bioimaging with WLI was demonstrated as a novel rapid bacterial culture assay with several advantageous capabilities. Fast nondestructive counting of colony-forming units in a culture and simultaneous measurement of bacterial growth rates and colony morphology with this method may be beneficial in research and clinical applications where current methods are either too slow or are destructive.

Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.

Isolation of some gram-negative bacterial species from naturally infected silver European eel fish (Anguilla anguilla linnaeus, 1758)

The aim of this study is to inform isolations of Shewanella putrefaciens, Aeromonas sobria and A. veronii from the silver European eel fish (Anguilla anguilla) caught from the Gulf of Antalya, Turkey. Three silver eel fish samples as freshly dead (mean weight 75 g) were obtained by a local fisherman. For bacteriological study, inoculations from the head kidney, spleen and liver were made and inoculated onto Brain Hearth Infusion Agar (BHIA-S) supplemented with 1.5 % NaCl. The inoculated petri dishes were incubated at 24 ± 2°C for 72 hours. The end of the incubation period, bacterial colonies were subcultured. For presumptive identification of the bacterial strains, the strains were identified using a variety of phenotypic characteristics. The further identification of the strains was performed with PCR. Clinically, the silver eel samples showed pale gills, losses of scale, necrotic lesions on dorsal, hemorrhagies on the jaws, lateral and ventral sides of the body and around the anus and hemoragic anal fin. At necropsy, the fish had enlarged and pale spleen and the gastrointestinal tract was empty. After 72 hours, 10 bacterial strains were isolated. 6 strains as Shewanella putrefaciens, 2 strains as Aeromonas sobria and 2 strains as A. veronii were identified according to the phenotypical characteristics and PCR study results. The eel fish stocks in natural conditions are decreasing and this problem may be multifactorial such as disease, migration and overhunting. As a result of this study, A. veronii was first isolated from A. anguilla.