Luria-Bertani Research Articles

Antimicrobial efficacy of photodynamic therapy, diode laser, and sodium hypochlorite and their combinations on endodontic pathogens.

Sterilization of the root canal system is of prime importance for a successful root canal therapy. Lasers and photodynamic therapy (PDT) have become the latest choice to eradicate microorganisms in the root canal. To evaluate and compare the antimicrobial activity of diode laser, photodynamic therapy, and sodium hypochlorite along with their combinations on endodontic pathogens: Enterococcus faecalis and Streptococcus mutans. A total of 120 uniradicular teeth were stored in 5.2% NaOCl solution to remove organic residues and the crowns were sectioned at the cemento-enamel junction (CEJ) to obtain root canal length of 15 mm. Canals were shaped using step-back technique up to #40 K-file and were autoclaved. The selected teeth (specimen) were randomly divided into two equal groups with sixty teeth being inoculated with E. faecalis (Group E) and remaining sixty teeth with S. mutans (Group S). Further, the groups were subdivided according to the disinfection technique used. Ten teeth from each subgroup were disinfected with a diode laser, photo activated disinfection (PAD), sodium hypochlorite, a combination of sodium hypochlorite and diode laser, a combination of sodium hypochlorite and photo activated disinfection respectively. Ten teeth in each group served as control without any disinfection. The treated specimens were transferred to test tubes containing 5 ml sterile Luria Bertani broth, incubated and the bacterial count, optical density in each root specimen was calculated and compared. The pairwise comparison of colonies across the subgroups was done by the Kruskal-Wallis test and within the subgroups was carried out using the Mann-Whitney U test. The statistical implication was tested at 5% and the study was performed using SPSS 18.0 ver. (SPSS Inc.). A significant reduction (98%) in the E. faecalis count was observed when the NaOCl was used in combination with the diode laser or PAD. PAD along with 3% NaOCl presents the advantage of utilizing a lower wavelength laser beam. Hence, PAD in combination with NaOCl can be an alternative and better option for root canal disinfection for both the endodontic pathogens, E. faecalis and S. mutans.

Non-molecular characterization of pellicle formation by poultry Salmonella Kentucky strains and other poultry-associated Salmonella serovars in Luria Bertani broth

There is limited research concerning the biofilm-forming capabilities of Salmonella Kentucky, a common poultry isolate. The objective was to quantitate pellicle formation of S. Kentucky versus better-characterized Salmonella strains of Enteritidis and Heidelberg. In separate experiments, Salmonella strains and serovars were tested for their biofilm-forming abilities in different Luria-Bertani (LB) broths (1); pellicle formation in different volumes of LB without salt (2); and the potential priming effects on formation after pellicles were transferred three consecutive times (3). Data were analyzed using One-Way ANOVA with means separated using Tukey’s HSD (P ≤ 0.05). In the first experiment, there was no significant effect between strain and serovars (P > 0.05), but media type affected pellicle formation significantly with LB Miller and LB minus NaCl plus 2% glucose resulting in no pellicle formation (P < 0.001). When grown in 50 mL, Kentucky 38-0085 produced larger pellicles than Kentucky 38-0055, and Heidelberg strain 38-0127 (P < 0.0001). Serial transfers of pellicles did not significantly affect pellicle formation (P > 0.05); however, Kentucky 38-0084, 38-0085 and 38-0086 produced larger pellicles than Kentucky 38-0055 and 38-0056 and Heidelberg 38-0126, 38-0127 and 38-0152. The current study demonstrates the consistent biofilm forming capabilities of Kentucky and may explain why Kentucky is frequently isolated in poultry processing facilities.

Biofilm production under different atmospheres and growth media by Streptococcus agalactiae isolated from milk of cows with subclinical mastitis

Different methods to analyze Streptococcus agalactiae biofilm formation have been investigated, but standardized protocols have not been developed. We compared S. agalactiae biofilm production among different atmospheres and growth media. Biofilm formation was studied in 32 isolates from bovine mastitis cases grown in Tryptone Soy Broth (TSB), Todd Hewitt Broth (THB), Luria Bertani Broth (LB) and Brain Heart Infusion (BHI), under two atmospheres, aerobic and 5% CO2. Regardless of the culture medium, growth under 5% CO2 resulted in a greater proportion of biofilm formation (65.63%), as compared with aerobic conditions (39.84%). Regardless of the atmosphere, the chances of biofilm formation were greater for isolates grown in TSB, as compared with THB [Odds ratio (OR) = 3.02], BHI (OR = 4.57), or LB (OR = 10.20). Thus, we suggest the use of 5% CO2 atmosphere and TSB in biofilm formation assays by Group-B streptococci (GBS) isolated from intramammary infections.

Influence of two different culture media on biofilm formation by Listeria monocytogenes isolated from a small-scale meat processing facility

In this study, 20 Listeria monocytogenes isolates detected in a food processing environment and food products were tested for biofilm-forming ability in two different culture media: Tryptone Soya Broth and Luria Bertani Broth. Statistical analysis of the data obtained was performed with the MINITAB software package, version 16.0. The two-sample t-test and confidence interval were used for data analysis. Significant differences between the isolates were observed in the ability to form biofilms.

Microbial Pb(II)-precipitation: the influence of oxygen on Pb(II)-removal from aqueous environment and the resulting precipitate identity

The study aimed to quantify the lead(II) bio-precipitation effectiveness, and the produced precipitate identities, of industrial consortia under aerobic and anaerobic batch conditions. The consortia were obtained from an automotive battery recycling plant and an operational lead mine in South Africa. The experiments were performed in the complex growth medium Luria–Bertani broth containing 80 ppm lead(II). The precipitation and corresponding removal of lead(II) were successfully achieved for both aerobic (yellow/brown precipitate) and anaerobic (dark grey/black precipitate) conditions. The removal of lead(II) followed similar trends for both aeration conditions, with the majority of lead(II) removed within the initial 48 h, followed by a marked decline in removal rate for the remainder of the experiments. The final lead(II) removal ranged between 78.11 ± 4.02% and 88.76 ± 3.98% recorded after 144 h. The precipitates were analysed using XPS which indicated the presence of exclusively PbO and elemental lead in the aerobic precipitates, while PbO, PbS, and elemental lead were present in the anaerobic precipitates. The results indicated an oxidation–reduction mechanism with lead(II) as an electron acceptor in both aerobic and anaerobic conditions, while a sulphide-liberation catabolism of sulphur-containing amino acids was evident exclusively in the anaerobic runs. This study provides the first report of bacterial bio-reduction in aqueous lead(II) to elemental lead through a dissimilatory lead reduction mechanism. It further provides support for the application of bioremediation for the removal and recovery of lead from industrial waste streams through the application of bacterial biocatalysts for direct elemental lead recovery.

Repeated in-vitro and in-vivo exposure leads to genetic alteration, adaptations, and hypervirulence in Salmonella

Adaptation is an important phenomenon for the survival of any organism in various in-vitro and in-vivo conditions leading to their better survival. We studied Salmonella, for the evolutionary adaptation under different conditions. When Salmonella was passaged for 12 rounds in Luria Bertani (P12-STM) media, F media (F12-STM) and Caenorhabditis elegans (Ce12-STM), we found certain phenotypic variations with respect to unpassaged strain. The passaged strains showed a significant increase in motility, body size and number of flagella they possessed, increased tolerance to ROS and RNS under in-vitro condition. They were more invasive in the human intestinal epithelial cells (Int-407) and showed a higher proliferation rate in the phagocytic cells (U-937 and RAW-264.7) suggesting their efficient intracellular proliferation. The evolved strains were hyper-infectious and reduced host viability significantly in C. elegans. The upregulation of various virulent and regulatory genes observed could be associated with the evolved adaptive virulent phenotype by Salmonella in a given condition. Whole genome sequencing also indicates the genetic alteration including insertion and deletions, single nucleotide polymorphisms which are common in all the strains as well as exposure specific, giving varied phenotype among the passaged strains. This study indicates that Salmonella is showing adaptation, genetic alteration and have become hypervirulent hence is capable of causing disease severity.

Isolation and Molecular Characterization of Acid Producing Bacteria from Selected Oilfield Environments within the Niger Delta

Acid producing bacteria are considered an important group of corrosive bacteria that have economic importance to petroleum industry. In this research, acid producing bacteria were isolated from produced water sample collected at ten (10) oil field environments within the Niger Delta region. The multiple tube fermentation technique was used to isolate the bacteria while Phenol red dextrose broth was used as the microbiological medium for the isolation of the acid producing bacteria. Also total heterotrophic bacteria count (THBC) was determined under aerobic and anaerobic condition using the standard plate count technique and the boiling method used for the extraction of acid producing bacterial DNA after growing in Luria Bertani broth. The extracted bacterial DNA were purified and quantified before PCR amplification. The PCR amplicons were subjected to gel electrophoresis. The bacterial DNA bands were quantified using 1500bp ladder. The result obtained showed that some acid producing bacteria isolated could survive as facultative microorganisms belonging to genera such as Klebsiella, Pantoea, Escherichia, Providentia, Proteus, Shewanella, Myroides and Pseudomonas. There was growth in all samples under aerobic condition with a THBC ranging from 3.602x102Cfu/ml – 4.698x102Cfu/ml while the range was within 3.301x102Cfu/ml – 5.676x102Cfu/ml under anaerobic condition. For physicochemical parameters determined, temperature range for all samples was within 23.9ºC – 24.8ºC; pH was within 7.24 – 8.10; total dissolved solids was within 470 mg/ml – 16160 mg/ml and conductivity was within 1.885 µs/cm – 845.2 µs/cm. The results also showed that acid producing bacteria grow mostly under aerobic condition unlike the sulphate reducing bacteria.

Multimetal Resistant, Alkalitolerant Bacteria Isolated from Serpentinizing Fluid-Associated Sediments and Acid Mine Drainage in the Zambales Ophiolite, the Philippines

Weathering in ophiolitic exposure settings often produces fluids and sediments that have elevated concentrations of various toxic heavy metals such as chromium, nickel, cobalt, copper, and zinc. Microorganisms inhabiting these environments (e.g., serpentinite soils or sediments) are likely adapted to tolerate these metals along with other physicochemical extremes. The purpose of this study is to isolate bacteria capable of tolerating extremely high concentrations of multiple metals and to assess the various tolerance mechanisms exhibited by different organisms. Sediment samples for microbial culturing were collected from Manleluag Spring National Park and Barlo Mine located on the island of Luzon, the Philippines. Luria-Bertani (LB) agar medium was supplemented with increasing concentrations of five trace elements – Cu, Cr, Co, Ni, and Zn. Over 20 isolates were obtained from media with concentrations ranging from 25 mg/L to 400 mg/L of each metal. Most isolates were identified as belonging to the genus Bacillus. Adaptation mechanisms, including potential biomineralization, were considered for select strains using inductively coupled plasma optical emission spectroscopy (ICP-OES), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). ICP-OES analysis suggests that the primary resistance mechanism used by most isolates is cellular efflux, although it was determined that some organisms were able to reduce aqueous Cu concentration by up to 40%. Most strains exhibited growth at pH 12, demonstrating that alkalitolerant, highly metal resistant organisms are found in these ophiolite-associated environments. These organisms may be exploited for bioremediation, secondary metabolite production, and other industrial applications.

Arthrobacter terricola sp. nov., isolated from forest soil.

A Gram-stain-positive, aerobic and rod-shaped bacterial strain, designated JH1-1T, was isolated from a forest soil sample collected in Suwon, Gyeonggi-do, Republic of Korea. Strain JH1-1T could grow at 10-35 °C (optimum, 28-30 °C), pH 4.5-8.5 and tolerated 5 % (w/v) NaCl. Strain JH1-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter alkaliphilus LC6T (98.5 % similarity), Arthrobacter methylotrophus TGAT (98.4 %), Arthrobacter ramosus CCM 1646T (97.8 %), Arthrobacter bambusae THG-GM18T (97.5 %) and Arthrobacter pokkalii P3B162T (97.3 %). The strain grew well on Reasoner's 2A agar, tryptone soya agar, nutrient agar, Mueller-Hinton agar and Luria-Bertani agar. The major polar lipid profile comprised phosphatidylglycerol, diphosphatidylglycerol, unidentified phospholipid and unidentified glycolipids. The major respiratory quinone was MK-9(H2). The main fatty acids were C15 : 0 anteiso, C15 : 0 iso, C16 : 0 iso and C17 :0 anteiso. The DNA G+C content of the isolated strain based on the whole genome sequence was 63.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain JH1-1T and its reference type strains ranged from 81.3 to 85.4 % and from 21.1 to 29.1 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, strain JH1-1T could be differentiated phylogenetically and phenotypically from the recognized species of the genus Arthrobacter. Therefore, strain JH1-1T is considered to represent a novel species, for which the name Arthrobacter terricola sp. nov. is proposed. The type strain is JH1-1T (=KACC 21385T=JCM 33641T).

Antifungal spectrum characterization and identification of strong volatile organic compounds produced by Bacillus pumilus TM-R

To obtain biocontrol agents for suppression of food-deteriorating fungi during storage of agricultural products, bacteria producing volatile organic compounds (VOCs) with strong antifungal activity were screened and isolated from various environmental samples. Among 136 bacterial isolates, strain TM-R showed the strongest and broadest antifungal activity. Based on physiological and genetical characterization, the bacterium was identified as Bacillus pumilus. The effects of VOCs produced by the bacterium, which was grown on four types of agar media (nutrient, Trypto-Soya, Luria–Bertani, and TM Enterprise), were examined against six species of fungi (Alternaria alternata, Aspergillus niger, Cladosporium cladosporioides, Curvularia lunata, Fusarium oxysporum, and Penicillium italicum) in both small- and large-scale tests (plate and 12-L tests, respectively). In the plate test, the bacterium markedly suppressed the mycelial growth of five fungi (Alternaria alternata, Cladosporium cladosporioides, Curvularia lunata, F. oxysporum, and P. italicum) but promoted the growth of Aspergillus niger. In the 12-L test, the degree of growth inhibitiondecreased; however, the bacterium grown on TMEA still exhibited the strongest inhibition, especially against P. italicum (growth inhibition rate of 93%). Surprisingly, the growth of Aspergillus niger was promoted even more strongly (−36%) by the bacterium on TMEA than in the plate test (−9%). Twenty-two of 32 VOCs detected by GC-MS were identified using three databases (NIST 2011, AromaOffice, and AroChemBase). The species and concentration of detected VOCs differed greatly among growth media. To identify causative antifungal VOCs, we estimated the correlation between growth inhibition of P. italicum by the bacterium grown on each of the four media vs. the relative abundance of individual VOCs. As a result, four VOCs (methyl isobutyl ketone, ethanol, 5-methyl-2-heptanone, and S-(−)-2-methylbutylamine) were determined to be the predominant antifungal VOCs. To the best of our knowledge, this study is the first to specify causative antifungal VOCs using such an approach.

Purification and cytotoxic assays of four antimicrobial metabolites extracted from Actinomycetes of the soils of Menengai Crater, Kenya

The search for antibiotic metabolites is increasing due to drug resistance been witnessed today. This study was conceived to isolate, identify and compare the antimicrobial metabolites yield of four actinomycetes isolated from Menengai crater soil, Kenya. The actinomycetes were isolated using starch casein, Luria Bertani (M1) and starch nitrate agar. The antimicrobial metabolites were extracted using standard techniques followed by purification, test for antagonism against selected pathogenic microbes, and cytotoxicity assay using Brine shrimp lethality test. Totally, 138 actinomycetes isolates were obtained from all the soil samples. Four isolates showed the highest potent potential against selected bacterial and fungal pathogens. The selected actinomycetes were coded; PAN 30, PAN 37, PAN 41, and PAN 154, and preserved at 4°C for further analysis. The difference in yield of the antimicrobial metabolites between liquid and solid-state fermentation was statistically significant (P= 0.005). Zones of inhibition did not vary significantly (F = 6.6046, P = 0.001338). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) of the antimicrobial metabolites extracted from PAN 30, PAN 37, PAN 41 and PAN 154 isolates were equal. The Half Lethal Concentration (LC50) in (µg/ ml) for the antimicrobial metabolites extracted from the 4 isolates were; PAN 30 (1.8168 ± 0.47), PAN 37 (3.4269 ± 0.56), PAN 41 (3.4269 ± 0.45), and PAN 154 (4.9397 ± 0.41). The four actinomycetes produced bioactive metabolites. Solid-state fermentation was superior to liquid-state fermentation in recovering the antimicrobial metabolites from the actinomycetes isolates. Molecular identification of the selected isolates needs to be carried out in a future study. Moreover, there is a need to determine the structure of the antimicrobials using nuclear magnetic resonance.

Oleisolibacter albus gen. nov., sp. nov., isolated from an oil-contaminated soil.

A Gram-stain-negative, aerobic, flagellated, rod-shaped bacterium, designated strain NAU-10T, was isolated from an oil-contaminated soil collected in PR China. Strain NAU-10T could grow at 10-42 °C (optimum, 30 °C), pH 5.0-9.0 (pH 7.0) and in the presence of 0-2.5 % (w/v) NaCl (0.5 % in Luria-Bertani broth). The major fatty acids were C18 : 1 ω7c (38.6 %), C17 : 1 ω6c (9.8 %), C18 : 1 2-OH (9.1 %), summed feature 3 (8.7 %), C16 : 0 3-OH (7.2 %) and C16 : 0 (6.7 %). The major respiratory quinones were Q9 and Q10. The total polar lipids were lipid, aminolipid, phospholipid, phosphatidylglycerol and phosphatidylethanolamine. Strain NAU-10T shared the highest 16S rRNA gene sequence similarities with Rhodocista pekingensis 3-pT (95.9 %), Niveispirillum cyanobacteriorum TH16T (95.3 %) and Niveispirillum fermenti CC-LY736T (95.3 %), and constituted a sub-cluster within the family Rhodospirillaceae. The DNA G+C content of strain NAU-10T was 68.2 mol% based on its draft genome sequence. Genome annotation of strain NAU-10T predicted the presence of 4309 genes, of which 4237 are coding proteins and 72 are RNA genes. Based on its phenotypic and chemotaxonomic characteristics, as well as the analysis of the 16S rRNA gene sequences, it was concluded that strain NAU-10T represents a novel genus, for which the name Oleisolibacter gen. nov., is proposed. The type species of this genus is Oleisolibacter albus with the type strain NAU-10T (=KCTC 62417T=CCTCC AB 2018015T).

Characterization and Initial Application of Endophytic Bacillus safensis Strain ZY16 for Improving Phytoremediation of Oil-Contaminated Saline Soils.

Hydrocarbon-degrading and plant-growth-promoting bacterial endophytes have proven useful for facilitating the phytoremediation of petroleum-contaminated soils with high salinity. In this study, we identified Bacillus safensis strain ZY16 as an endophytic bacterium that can degrade hydrocarbons, produce biosurfactants, tolerate salt, and promote plant growth. The strain was isolated from the root of Chloris virgata Sw., a halotolerant plant collected from the Yellow River Delta. ZY16 survived in Luria-Bertani (LB) broth with 0–16% (w/v) sodium chloride (NaCl) and grew well in LB broth supplemented with 0–8% NaCl, indicating its high salt tolerance. The endophytic strain ZY16 effectively degraded C12–C32 n-alkanes of diesel oil effectively, as well as common polycyclic aromatic hydrocarbons under hypersaline conditions. For example, in mineral salts (MS) liquid medium supplemented with 6% NaCl, ZY16 degraded n-undecane, n-hexadecane, n-octacosane, naphthalene, phenanthrene, and pyrene, with degradation percentages of 94.5, 98.2, 64.8, 72.1, 59.4, and 27.6%, respectively. In addition, ZY16 produced biosurfactant, as confirmed by the oil spreading technique, surface tension detection, and emulsification of para-xylene and paraffin. The biosurfactant production ability of ZY16 under hypersaline conditions was also determined. Moreover, ZY16 showed plant-growth-promoting attributes, such as siderophore and indole-3-acetic acid production, as well as phosphate solubilization. To assess the enhanced phytoremediation of saline soils polluted by hydrocarbons and the plant-growth-promotion ability of ZY16, a pot trial with and without inoculation of the endophyte was designed and performed. Inoculated and non-inoculated plantlets of C. virgata Sw. were grown in oil-polluted saline soil, with oil and salt contents of 10462 mg/kg and 0.51%, respectively. After 120 days of growth, significant enhancement of both the aerial and underground biomass of ZY16-inoculated plants was observed. The soil total petroleum hydrocarbon degradation percentage (a metric of phytoremediation) after incubation with ZY16 was 63.2%, representing an elevation of 25.7% over phytoremediation without ZY16 inoculation. Our study should promote the application of endophytic B. safensis ZY16 in phytoremediation by extending our understanding of the mutualistic interactions between endophytes and their host plants.

Expression and characterization of recombinant human lactoferrin in edible alga Chlamydomonas reinhardtii.

Lactoferrin (LF) is a naturally occurring iron-binding glycoprotein with a variety of biological functions. It has increasing demand every year and huge market potential. In this study, we explored the feasibility of expressing human LF (hLF) in edible algae C. reinhardtii. A codon-optimized hLF gene was synthesized, inserted into pCAMBIA-1301C and transformed into C. reinhardtii SP strain. In total, 7 hLF-expressing clones were selected with clone 121 exhibiting the highest expression level. The hLF-containing algal extract significantly inhibited the growth of bacteria such as Escherichia coli and Klebsiella variicola. During acute toxicity experiment no acute toxicity was detected, especially on changes of the body weight and histopathology of organs. The recombinant hLF possessed a similar or modestly reduced stability compared to commercial hLF standard. Our data indicated that expression of hLF in C. reinhardtii is feasible and paved a way to commercial production of lactoferrin using edible Chlamydomonas expression system. Abbreviations: atrazine chlorohydrolase gene (atzA); bovine serum albumin (BSA); human LF (hLF); lactoferrin (LF); Luria-Bertani (LB); quantitative reverse transcriptase PCR (qRT-PCR) ; SDS polyacrylamide gel electrophoresis (SDS-PAGE); Tris-acetate phosphate (TAP); western blotting (WB).

Potential application of a recombinant bacterial strain carrying a groEL promoter as a whole-cell microbial biosensor for detecting bioavailable cadmium

Abstract In this study, we investigated the response to heavy metals of recombinant E. coli NovaBlue strains carrying plasmids harboring different Cupriavidus taiwanensis LMG19424-derived groEL promoter fragments fused to the lacZ reporter gene. Four recombinant plasmids (designated G14, G26, G27 and G28) were transformed into E. coli and screened for the ability to indicate the presence of heavy metals on Luria–Bertani (LB) agar. Only the G28 biosensor exhibited reproducible results, with an observed limit of Cd detection of 3 μ g/mL. In LB broth, the NovaBlue host strain was able to grow and tolerate in the presence of 50 μ g/mL As, Cd, Co, Ni, Pb and Zn but was susceptible to 6.2 μ g/mL Cr. The β -galactosidase ( β -gal) reporter activity was reduced when the concentration of heavy metals increased, with observed detection limits, as indicated by the blue color, of 25 μ g/mL for CdCl2, CdSO4, and CdCO3; 50 μ g/mL for As (III) and ZnSO4; and 100 μ g/mL for CoCl2 and NiSO4. The specificity of the induction ratio and metal concentration was highest for Cd, with R2 values of 0.9196 and 0.9164 for CdCl2 and CdSO4, respectively. When the ratio of β -gal activity and growth was compared between NovaBlue strains carrying G28 or pETBlue-2 (containing an E. coli promoter), the G28 biosensor exhibited a significantly higher ratio than the strain carrying pETBlue-2 at 0.8–6.3 μ g/mL CdCl2. The results demonstrated that the certain concentration range corresponded to Cd induction of G28. The G28 biosensor were immobilized on paper discs and tested using the most toxic and soluble Cd compound (CdCl2) at 0–0.8 μ g/mL on LB agar. The G28 biosensor developed a blue color only in the absence of CdCl2. However, when testing soil extracts contaminated with low, medium or high levels of total Cd or those spiked with CdCl2 (up to 40 μ g/mL), all the G28 biosensor discs showed a similar growth pattern with a spotted blue color compared with the even blue color of the control disc. Due to the complexity of environmental samples, several factors may interfere with the metal response of the G28 biosensor. However, the G28 biosensor exhibited different behaviors in the presence of specific types and concentrations of metals. Thus, combinations of G28 and more specific promoters should be further investigated to improve the sensitivity and specificity of the groEL promoter for detecting Cd and other heavy metals in contaminated samples.

Poly(vinyl pyrrolidone)-mediated synthesis of silver nanowires decorated with silver nanospheres and their antimicrobial activity

Pathogenic infectious diseases, like bacterial infections, are one of the most prevalent types of diseases all over the world. Although antibiotics are commonly used and widely available drugs for the treatment of bacterial infections, bacteria show multiple drug resistance, have a tendency to get genetically mutated and become resistant to commonly employed antibiotic drugs. This makes the invention of novel drugs essential. One of the possible approaches is the use of nanomaterials for this purpose. The present study attempts to synthesize silver nanospheres and silver nanowires and compare their antibacterial activity. Silver nanospheres were synthesized by chemical reduction of silver nitrate in the presence of a suitable stabilizer. For the synthesis of silver nanowires, polyol method was employed. Silver nanowires neatly decorated with silver nanospheres were obtained. The antibacterial activity was estimated by separately determining the minimum inhibition concentration of the two nanostructures on Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli cultured on Luria Bertani agar media. The comparison shows that the antibacterial activity of silver nanospheres is better than that of silver nanowires which is attributed to its higher surface area and the difference in uptake mechanism by the bacterial cells.

From beta-cyclodextrin polyelectrolyte to layer-by-layer self-assembly microcapsules: From inhibition of bacterial growth to bactericidal effect

Pathogenic bacteria resistance to antimicrobial agents has emerged as a major health issue. Developing new systems enabling inhibitory and bactericidal effects is crucial. Herein, layer-by-layer technique (LbL) was used to fabricate microcapsules composed of biopolymers, cationic polycyclodextrin (P(CD+)) and anionic alginate (alg−). This was achieved through successive adsorption of these polyelectrolytes onto calcium carbonate microparticles (CaCO3) used as template. Then, a chelating agent was added for removing the CaCO3 core, leading to microcapsules. Quartz crystal microbalance and zeta potential measurements evidenced the successful alternate adsorption of polyelectrolytes and related charge reversal, respectively, upon the LbL deposition. The shape of the capsules was characterized by Scanning Electron Microscopy (SEM) and fluorescence microscopy. The antibacterial activity of P(CD+) was first evidenced on two well-known bacteria: Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) using well diffusion method on Plate Count Agar (PCA) medium. Then, an inhibitory effect on the bacteria growth has been observed for the P(CD+)-based microcapsules during bacterial culture in Luria-Bertani (LB) broth medium. Our results pointed out towards a strong dependence of the extent of inhibition on the number of self-assembled polyelectrolytes' layers and capsules’ concentration. The effect increased with both concentration and number of layers. Acridine orange staining evidenced the bactericidal effect of P(CD+)-based microcapsules. This contribution demonstrates unambiguously that: i) aqueous solutions of P(CD+) exhibit antibacterial activity at physiological pH suggesting that P(CD+) can act as a versatile alternative to the widely studied chitosan whose antimicrobial activity is limited to acidic pH and ii) (P(CD+)/alg−)n hollow capsules can be exploited as an antibacterial agents with additional host properties.

Salmonella enterica Growth Conditions Influence Lettuce Leaf Internalization.

Human pathogens on plants (HPOP) have evolved complex interactions with their plant host. Stomatal internalization is one such mode of interaction, where bacteria are attracted to stomata and penetrate into the substomatal cavity by a process mediated by chemotaxis. Internalization enables HPOP to evade the hostile environment of the leaf surface and find a protected, nutrient-rich niche within the leaf. Numerous studies have documented attachment and entry of the foodborne pathogens, Salmonella enterica and Escherichia coli into stomata. Internalization, however, varies considerably among different pathogens and in different plants, and both bacterial and plant’s factors were reported to influence HPOP attachment and internalization. Here we have studied the effect of laboratory growth conditions, on the internalization of Salmonella enterica serovar Typhimurium (STm) into lettuce leaf. We have further tested the potential involvement of universal stress-proteins in leaf internalization. We found that STm grown in Luria Bertani broth devoid of NaCl (LBNS), or in diluted LB (0.5×LB) internalized lettuce leaf better (62 ± 5% and 59 ± 7%, respectively) compared to bacteria grown in LB (15 ± 7%). Growth under non-aerated conditions also enhanced STm internalization compared to growth under aerated conditions. Growth temperature of 25 and 37°C did not affect STm internalization, however, growth at 42°C, significantly augmented leaf internalization. Since, the tested growth conditions represent moderate stresses, we further investigated the involvement of five universal-stress genes in STm leaf internalization following growth in LBNS medium. Knockout mutations in ydaA, yecG, ybdQ, and uspAB, but not in ynaF, significantly reduced STm internalization compared to the wild-type (wt) strain, without affecting bacterial attachment and motility. Transduction of the mutations back to the parent strain confirmed the linkage between the mutations and the internalization phenotype. These findings support a specific role of the universal-stress genes in leaf internalization. The present study highlights the complexity of bacterial internalization process and may provide partial explanation for the variable, sometimes-contrasting results reported in the literature regarding stomatal internalization by HPOP. Characterization of the regulatory networks that mediate the involvement of usp genes and the tested growth factors in STm internalization should contribute to our understanding of human pathogens-plant interactions.

Antimicrobial peptide KR-32 alleviates Escherichia coli K88-induced fatty acid malabsorption by improving expression of fatty acid transporter protein 4 (FATP4)1.

Bacterial infection causes nutrient malabsorption in small intestine. KR-32, a kind of synthetic antimicrobial peptide, has the bacteriostatic effect. In the present study, 2 experiments were designed to analyze the effects of KR-32 on fat absorption of piglets with or without Escherichia coli infection. In Exp. 1, 12 weaning piglets (21 d old) were allocated to 2 groups: piglets with an intraperitoneal (i.p.) injection of antimicrobial peptide KR-32 (APK) and piglets with an i.p. injection of an equivalent volume (1 mL) of phosphate-buffered saline (PBS) (CON-1). Results showed that after 7 d of growth, KR-32 did not significantly change growth performance and apparent total tract digestibility (ATTD) of feed nutrients of normal pigs. To confirm whether KR-32 affects those of enterotoxigenic Escherichia coli (ETEC) K88-challenged pigs, we performed Exp. 2, in which 18 piglets (28 d old) were divided into the following 3 groups: 1) piglets orally challenged with 1 × 1010 cfu ETEC K88 on day 1 followed by an i.p. injection of 0.6 mg/kg KR-32 (K88 + APK); 2) piglets orally challenged with 1 × 1010 cfu ETEC K88 on day 1 followed by an i.p. injection of an equivalent volume (1 mL) of PBS (K88); and 3) piglets with an oral administration of fresh Luria-Bertani broth (50 mL) followed by an i.p. injection of an equivalent volume of PBS (CON-2). Results showed that ETEC K88 challenge led to poor ADFI, ADG, and G:F in piglets; decreased ATTD of feed nutrients, especially CP and ether extract (EE); and intestinal morphology disorder. After i.p. injection of KR-32, ADG and ATTD of CP and EE were greatly increased, G:F was significantly reduced (P < 0.05), and, especially, ATTD of EE returned to a normal level compared with group CON-2. Fatty acid absorption also highly increased after KR-32 injection. Then we focused on fat digestion and fatty acid uptake. The pH in the intestine and pancreas lipase showed no difference among the 3 treatment groups, whereas fatty acid transporter protein 4 (FATP4) expression was remarkably improved (P < 0.05) and the epithelial barrier was recovered after i.p. injection of KR-32. In conclusion, KR-32, given to ETEC K88-challenged piglets, improved growth performance, ATTD of EE, fatty acid absorption, and intestinal morphology, which indicated that KR-32 was likely to improve the expression of FATP4 and by repairing the epithelial barrier, thereby alleviating fatty acid malabsorption.

Effect of the spoIIID mutation on mother cell lysis in Bacillus thuringiensis.

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A β-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.