Bacterial Strain Typing Research Articles

Microwave induced synthesis, and pharmacological properties of novel 1-benzoyl-4-bromopyrrolo[1,2-a]quinoline-3-carboxylate analogues

Abstract Eco-friendly methods of heterocyclic synthesis are highly important in view of pharmacology. This synthetic chemistry deals with the application of environmental friendly chemical compounds synthesis using microwave methods. Greener method of synthesis of benzo-fused indolizines like ethyl 4-Bromo-1-(substituted-benzoyl)pyrrolo[1,2-a]quinoline-3-carboxylate analoguos 1a-n using the one-pot method with 3‑bromo quinolone, phenacyl bromide and electron-deficient alkynes in the presence of triethylamine base with acetonitrile as a solvent. The structures of all the newly synthesized pyrrolo[1,2-a]quinoline have been confirmed by spectroscopic techniques. All the synthesized test compounds were screened for their antibacterial activity using six different types of gram-positive and gram-negative bacterial strains. The compounds 1a, 1k, and 1l exhibited a significant zone of inhibition against all six bacterial strains, and the test compound 1i showed potent free radical scavenging activity with the highest percentage inhibition of 49.27 at 100 µg/mL.

Impact of different transition metal ions in the structural, mechanical, optical, chemico-physical and biological properties of nanohydroxyapatite

Pure and metal ions (iron, manganese, cobalt) doped nanosized hydroxyapatite [Ca10(PO4)6(OH)2 or HAP] were prepared by wet-chemical precipitation technique. XRD patterns confirm that all the synthesized samples are single phase HAP. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) clearly showed the hexagonal and spherical morphology of the samples. X-ray photoelectron spectroscopy (XPS) confirmed that Fe2+, Mn2+, and Co2+ are substituted in HAP separately, and it also confirmed their oxidation state. The ac conductivity increases with the rise in frequency for all the samples. Among all dopants, 1% Fe doped HAP shows both the highest ac conductivity and the lowest impedance. The ac conductivity of the samples is in the order of 10−6 S/m, and it remains the insulating nature of the pure and doped HAP. The diamagnetic nature of the HAP changed into superparamagnetic due to the addition of dopant. The antibacterial activity of chosen samples depends on the types of bacterial strains and the dopant concentration. Among the different dopants 5% cobalt doped HAP revealed the highest antibacterial activity against Shigella dysenteriae bacteria.

Necrotizing enterocolitis is preceded by increased gut bacterial replication, Klebsiella, and fimbriae-encoding bacteria.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease that occurs primarily in premature infants. We performed genome-resolved metagenomic analysis of 1163 fecal samples from premature infants to identify microbial features predictive of NEC. Features considered include genes, bacterial strain types, eukaryotes, bacteriophages, plasmids, and growth rates. A machine learning classifier found that samples collected before NEC diagnosis harbored significantly more Klebsiella, bacteria encoding fimbriae, and bacteria encoding secondary metabolite gene clusters related to quorum sensing and bacteriocin production. Notably, replication rates of all bacteria, especially Enterobacteriaceae, were significantly higher 2 days before NEC diagnosis. The findings uncover biomarkers that could lead to early detection of NEC and targets for microbiome-based therapeutics.

Read-SpaM: assembly-free and alignment-free comparison of bacterial genomes with low sequencing coverage

BackgroundIn many fields of biomedical research, it is important to estimate phylogenetic distances between taxa based on low-coverage sequencing reads. Major applications are, for example, phylogeny reconstruction, species identification from small sequencing samples, or bacterial strain typing in medical diagnostics.ResultsWe adapted our previously developed software program Filtered Spaced-Word Matches (FSWM) for alignment-free phylogeny reconstruction to take unassembled reads as input; we call this implementation Read-SpaM.ConclusionsTest runs on simulated reads from semi-artificial and real-world bacterial genomes show that our approach can estimate phylogenetic distances with high accuracy, even for large evolutionary distances and for very low sequencing coverage.

Exploring Therapeutic Potential of Atorvastatin Against Gram-positive and Gram-negative Bacteria: In silico, In vitro and In vivo Evidences.

The incidences of opportunistic bacterial infections have increased in the past two decades or is expected to increase in the near future. Despite the availability of various classes of antibiotics, bacterial infections are not handled properly. Thus, in the present study, we have repurposed atorvastatin against various types of bacterial strains by using in silico, in vitro, and in vivo studies. Furthermore, a preliminary safety study was conducted using MTT assay. In silico study results have revealed that atorvastatin has good interaction with various targets of bacterial cell like that of the reference ligand. However, under in vitro conditions,it was found that atorvastatin was effective at higher concentrations (>128 μg/ml) against various bacterial strains. Thus, further, atorvastatin was tested in combination with standard antibiotics and has shown a synergistic effect. The MTT assay results have revealed non-cytotoxic activity of atorvastatin. In conclusion, atorvastatin in combination with standard drugs could be developed as an antibacterial agent.

243. Transflection Fourier Transform Infrared Spectroscopy as a Real-Time Strain Typing Technique: A Vancomycin-Resistant Enterococcus faecium (VRE) Typing Prospective Study

BackgroundRapid bacterial strain typing for nosocomial outbreak surveillance is critical for timely outbreak detection and implementation of appropriate infection control protocols in hospitals. Pulsed-field gel electrophoresis (PFGE) remains the gold standard for strain typing, but it has the disadvantages of being time-consuming and costly. Transflection Fourier transform infrared (FTIR) spectroscopy is a nondestructive and reagent-free technique for rapid microbial identification and subspecies-level discrimination. The potential of employing transflection FTIR spectroscopy as a rapid, real-time typing technique was evaluated in the present study.MethodsTransflection FTIR spectra were acquired from vancomycin-resistant Enterococcus faecium (VRE) isolates obtained from rectal swabs (n = 36) of patients in 6 units at a Montreal hospital over a 3-month period and from environmental screening samples (n = 2). Upon confirmation as VRE using a transflection FTIR spectral database previously developed in our laboratory, isolates were further typed by unsupervised hierarchical cluster analysis and principal component analysis of the FTIR spectral data with the use of a feature selection algorithm.ResultsAnalysis of the FTIR data identified independent cases of VRE outbreak in 2 of 6 units; these outbreaks were confirmed retrospectively by PFGE. Based on the PFGE typing results for all 38 isolates included in this study, FTIR spectral analyses successfully identified 95% (n = 18) of isolates related to the outbreaks and 95% (n = 18) of non-outbreak-related isolates, resulting in a false-positive (n = 1), and a false-negative (n = 1), rate of 5%. Additionally, the two environmental isolates were identified as part of the outbreak from one of the outbreak-positive units.ConclusionThe results in this study indicate that transflection FTIR spectroscopy-based typing can be considered as an alternative typing technique to PFGE, providing real-time results to track the spread of antibiotic-resistant pathogens within hospitals. Furthermore, when combined with the use of a transflection FTIR spectral database, both identification and typing of an isolate can be achieved from a single spectral measurement, thereby reducing the time and cost required for outbreak investigation.Disclosures All authors: No reported disclosures.

Production of Non-Toxic Biosurfactant – Surfactin – Through Microbial Fermentation of Biomass Hydrolysates for Industrial and Environmental Applications

Abstract The economically viable large-scale production of the pure isoforms of the surfactin biosurfactants, involving bacterial – Bacillus subtilis – fermentation of biomass hydrolysate feedstock, relies on the types of bacterial strains, optimization of the fermentation processing parameters, differences in the composition of the carbon and nitrogen in the bacterial media, and the chromatography techniques used for isolation of the isoforms. Here, we biosynthesized the surfactin isoforms in their mixture forms through fermentation of biomass hydrolysates at 2 wt.% carbohydrate content. The surfactin isoforms were assessed for their surface-active properties and toxicity. The enzyme hydrolysates considered were from switchgrass, soyhull (fiber), alfalfa, and bagasse. The isoform mixtures obtained after fermentation of the hydrolysates and, glucose as a control, were concentrated using chromatography columns, and characterized for molecular weights (MWs) and relative distribution using LCMS. The isoform mixtures, obtained in different fermenters (5- and 15-L) and, for different hydrolysates, invariably constituted 5 isoforms with MWs as 992.6, 1006.6, 1020.6, 1034.6, 1048.6, 1062.6 m/z amu, with their relative proportions as 6, 24, 35, 24, and 10 weight % respectively. The surface tension values of all these isoforms, in the absence of electrolytes and at 12 ppt salinity, were similar: 37 (pH 6.5) and 31 (pH 9.5) mN/m. Furthermore, the emulsification index values for the isoforms were also similar: Dispersant-to-Oil ratio as 1:20. The LC50 for Gulf killifish, Fundulus grandis for these surfactin isoforms ranged between 10 and 20 mg/L; a microbially-produced surfactin variant FA-Glu (Fatty acid Glutamate) was least toxic with LC50 at ∼100 mg/L. Thus, the surfactin synthesis approach adopted here suggested that pure (>95 wt.%) non-toxic isoforms of surfactin biosurfactants can be produced in the forms of their mixtures with surface-active properties similar to those of the pure forms of the surfactin isoforms.

Role of Annealing Temperatures on Mechanical, Optical, Electrical and Magnetic Properties of Nanohydroxyapatite Biomaterial.

Nanosized hydroxyapatite [Ca10(Po₄)6(OH)₂ or HAp] was prepared by applying wet-chemical precipitation technique. Thermogravimetric and Differential Scanning Calorimetry (TG/DSC) analyses, showed that the prepared sample was stable up to 919.2 °C. X-ray diffraction (XRD) pattern showed that as-prepared and annealed samples are in hexagonal structure. The average crystallite size is found to be 39, 46, 51 and 65 nm for the as-prepared, 700, 800 and 900 °C annealed HAp respectively. The dislocation density, strain and surface area were decreased with increase in annealing temperature. FT-IR and Raman spectra showed both as-prepared and annealed samples having characteristic bands of HAp. FE-SEM and TEM images which depicted and confirmed the hexagonal structure of HAp. The optical band gap calculated from the UV absorption behavior of as-prepared and annealed at 700, 800 and 900 °C HAp are 3.86, 3.84, 3.54, and 3.03 eV respectively. As the annealing temperature increases, the conductivity decreases whereas the impedance increases. The ac conductivity of as-prepared and annealed samples are in the order of 10-5 Sm-1. The VSM analysis confirmed that both as-prepared and annealed samples of HAp are in diamagnetic nature. The antibacterial activity of apatite samples depend on the types of bacterial strains and also their activity changed with the crystallite size.

Cano-wgMLST_BacCompare: A Bacterial Genome Analysis Platform for Epidemiological Investigation and Comparative Genomic Analysis.

With the decreasing cost of next-generation sequencing, whole-genome sequence-based bacterial genome comparisons are expected to become a mainstream process in the public health domain. Extended multilocus sequence typing (MLST) methods are becoming increasingly popular for use in comparing bacterial genetic relatedness in epidemiological investigations. Several extended MLST schemes based on biological signatures have been reported. Among them, whole-genome MLST (wgMLST) has gradually become one of the most widely used approaches for bacterial strain typing. In addition to using bacterial typing, many researchers aim to identify differences in the genes of compared strains. Because these differences might provide insights into critical bacterial functions, such as virulence and pathogenicity, researchers usually study these genes that differ between strains. Hence, we designed a web service tool based on wgMLST-constructed tree topology coupled with the feature selection method to create the “canonical wgMLST (cano-wgMLST) tree.” The genes that differ between strains are shown at each split of the tree, thereby directly providing information for performing a comparative genomic analysis for each strain pair. We demonstrated that this web service tool could be operated efficiently on two datasets consisting of 22 Campylobacter jejuni isolates and 59 S. Heidelberg isolates, respectively. We imposed this tool on a designated web server, cano-wgMLST_BacCompare, to enable users to create a wgMLST tree and canonical wgMLST tree automatically from their uploaded bacterial genomes for not only epidemiological investigation but also comparative genomic analysis. Additionally, detailed information on how to use this service is provided. The cano-wgMLST_BacCompare is available at http://baccompare.imst.nsysu.edu.tw.

MALDI TOF MS – new possibilities in routine microbiological diagnostics

One of the problems of modern medicine is diagnosis, treatment and prophylaxis of infections caused by multidrug-resistant strains of bacteria. Fast and correct identification of these pathogens is of utmost importance, as it enables early implementation of effective therapy. Therefore, rapid, modern and affordable methods are of outstanding value as they make it possible to conduct a reliable analysis in a very short period of time. One of such techniques is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which in recent years is increasingly used in clinical microbiological laboratories. This method enables cheap, fast and reliable identification of microorganisms based on their protein profiles. At present the possibilities of use of MALDI-TOF MS method become more broad, e.g. for identification of microorganisms directly in positive blood culture samples, typing of bacterial strains in epidemiological investigation of an outbreak as well as detection of multidrug-resistant strains, including producers of carbapenemases.

Commonality of Multidrug-Resistant Klebsiella pneumoniae ST348 Isolates in Horses and Humans in Portugal.

Multidrug-resistant (MDR) Klebsiella pneumoniae is considered a major global concern by the World Health Organization. Evidence is growing on the importance of circulation of MDR bacterial populations between animals and humans. Horses have been shown to carry commensal isolates of this bacterial species and can act as human MDR bacteria reservoirs. In this study, we characterized an extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae sequence type (ST) 348 isolate from a horse, an ST reported for the first time in an animal, using next-generation sequencing. We compared it with six other MDR K. pneumoniae ST348 human isolates previously identified in health-care facilities in Portugal using a core genome multi-locus sequence typing approach to evaluate a possible genetic link. The horse isolate was resistant to most of the antimicrobials tested, including 3rd generation cephalosporins, fluoroquinolones, and aminoglycosides, and presented several antimicrobial resistance genes, including blaESBL. Twenty-one allele differences were found between the horse isolate and the most similar human isolate, suggesting a recent common ancestor. Other similarities were observed regarding the content on antimicrobial resistance genes, plasmid incompatibility groups, and capsular and somatic antigens. This study illustrates the relevance of the dissemination of MDR strains, and enhances that identification of these types of bacterial strains in both human and veterinary settings is of significant relevance in order to understand and implement combined control strategies for MDR bacteria in animals and humans.

Nature Helps: Toward Bioinspired Bactericidal Nanopatterns

AbstractDevelopment of synthetic bactericidal surfaces is a drug‐free route to the prevention of implant‐associated infections. Surface nanotopographies with specific dimensions have been shown to kill various types of bacterial strains through a mechanical mechanism, while regulating stem cell differentiation and tissue regeneration. The effective ranges of dimensions required to simultaneously achieve both aims are in the <200 nm range. Here, a nanoscale additive manufacturing (=3D printing) technique called electron beam induced deposition (EBID) is used to fabricate nanopillars with reproducible and precisely controlled dimensions and arrangements that are within those effective ranges (i.e. a height of 190 nm, a diameter of 80 nm, and an interspacing of 170 nm). When compared to the flat surface, the nanopatterned surfaces show a significant bactericidal activity against both Escherichia coli and Staphylococcus aureus (with respective killing efficiencies of 97 ± 1% and 36 ± 5%). Direct penetration of nanopatterns into the bacterial cell wall leads to the disruption of the cell wall and cell death. The more rigid cell wall of S. aureus is consistent with the decreased killing efficiency. These findings support the development of nanopatterns with precisely controlled dimensions that are capable of killing both Gram‐negative and Gram‐positive bacteria.

Synthesis, spectroscopic characterization and biological studies of nano-sized Co(II), Ni(II), Cu(II) and Zn(II) Schiff's base hybrids

A novel series of nano-sized Co(II), Ni(II), Cu(II) and Zn(II) hybrids has been synthesized using the template method, i.e. by the in situ reaction of oxazin–Schiff's base [(E)-3-(2-(4-(diethylamino)-2-mercaptobenzylidene)hydrazineyl)-2H-benzo[b][1,4]oxazin-2-one] (HL) derived from 4-(diethylamino)-2-mercaptobenzaldehyde with 3-hydrazineyl-2H-benzo[b][1,4]oxazin-2-one in the presence of Co(II), Ni(II), Cu(II), Zn(II) chloride in ethanol. The structures and geometry of the optained compounds have been described by using the different analytical and spectroscopic tools such as elemental analysis, UV-VIS, IR, MS, 1H NMR, ESR, CV, TGA, XRD, SEM, AFM, magnetic and molar conductivity measurements. IR spectral studies confirmed the coordination of the ligand to the metal ions through the thiol sulfur, azomethine nitrogen and carbonyl oxygen. The Schiff's base ligand acts as monobasic tridentate OSN donor. The geometrical structures have been found to be octahedral and tetrahedral. The molar conductance measurements of the nano-sized hybrids in DMF and DMSO correspond to be nonelectrolytic nature for all nano-sized metal (II) hybrids. The elemental analyses confirm 1 M: 2 L stoichiometry of the type [ML2] (M = Co(II), Ni(II), Cu(II) and Zn(II)). Electrochemical behaviour of nano-sized metal (II) hybrids were determined by cyclic voltammetry. Degradation pattern of the ligand HL and its nano-sized hybrids is shown by thermal studies. The activation of thermodynamic parameters are calculated using Coast-Redfern (CR) and Horowitz-Metzger (HM) methods. The data obtained from XRD, SEM, TEM and AFM images unequivocally asserted the nano-sized metal chelates. The percentage antioxidant activities are also more for complexes than the ligand. The investigated ligand and metal complexes were screened for their in-vitro antimicrobial activities against different types of fungal and bacterial strains. The resulting data assert on the inspected compounds as a highly promising bactericides and fungicides. The antitumor activities of all synthesized compounds were evaluated towards human liver Carcinoma (HepG2) cell line. DNA-binding, DNA cleavage and fluorescence properties of nano-sized hybrids with this ligand are reported.

First report of the new emerging global clone ST1193 among clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from Germany.

Sequence type 1193 (ST1193) is a new emerging global clone of Escherichia coli. The main goal of this study was to determine the prevalence and molecular characteristics of ST1193 among clinical isolates of extended-spectrum β-lactamase (ESBL)-producing E. coli from University Hospital of Erlangen, Germany. Between November 2015 and February 2016, all consecutive non-duplicate clinical E. coli isolates showing resistance to cefotaxime or ceftazidime were further analysed for ESBL production by the combined disk method. ESBL genes were identified by PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, MLST and whole-genome sequencing. ESBL production was confirmed in 51 isolates. The globally dominant ST131 occurred at a frequency of 37.3% (n=19). Major non-ST131 sequence types were ST38 (n=4; 7.8%), ST10 (n=3; 5.9%) and ST1193 (n=3; 5.9%). Among the ESBL-producing E. coli ST1193, two expressed CTX-M-14 and one expressed CTX-M-15 ESBL type. All three ST1193 isolates belonged to serogroup O75:H5, phylogroup B2, and harboured IncFIA and IncFIB plasmids and the virulence factors genes iha, sat, gad, vat and senB. Moreover, they showed ciprofloxacin resistance and exhibited a set of four conserved mutations defining quinolone resistance (gyrA S83L, gyrA D87N, parC S80I and parC L416F). This study revealed for the first time in Germany the occurrence of ST1193 among clinical isolates of ESBL-producing E. coli. Further national or regional multicentre studies are needed to assess the effective relevance of ESBL-producing E. coli ST1193 as a nosocomial pathogen in Germany.

Environmental fate descriptors for glycol-coated selenium nanoparticles: a quantitative multi-assay approach.

The application of selenium nanoparticles (Se-NPs) in the fields of biological and physical science is escalating very fast. Therefore, it is necessary to investigate the effects of Se-NPs for their prospective vulnerability in the environment. The present work involved the investigation of the toxicological responses of four different types of glycols, i.e., EG, DEG, TEG and PEG coated on Se-NPs by using a multi-assay approach. The influence of the surface-coated Se-NPs was examined over four different types of bacterial strains. Additional work was carried out with glycol-coated Se-NPs for observing their anti-oxidative activities. To achieve the objective of assessing the environmental fate of the chosen Se-NPs, they were tested for assessing their hydrogen peroxide free radical scavenging activity and total reduction behaviour. The in vitro stability of the surface-coated Se-NPs was investigated over various components of blood. It was observed from the data that 15% glycol-encapsulated Se-NPs were biocompatible with the chosen components. The sorption, degradation and aggregation parameters were investigated to describe the environmental fate of the surface-coated Se-NPs. The results from the multi-assay activities are meaningful for improving the perception about the ecological fate of Se-NPs with their probable threats and reducing the uncertainty about their use.

CHEMICAL CONSTITUENTS AND ANTIBACTERIAL ACTIVITY OF Prosopisfarcta (Fabacea) FRUIT FROM IRAQ-KURDISTAN REGION

The chemical constituents of di chloro methane and hexane extracts of Prosopis Farcta (Fabacea family)were studied by gas chromatography, mass spectrometry. Their relative concentrations were determined and both extracts contained 8 expected compounds. There were 7 compounds found in hexane extract which are: 1- methyl pyrrole (8.08%), alpha-ethyl aspartate (9.96%), spiropentane  propyl (13.75%), ethyl oleate (43.43%), 1-iodo-n-Nonyliodide (7.6%), Borane-diethyl (decyloxy) (9.34%), Sulfurous acid, 2-ethyl hexyl ester (7.84%). While one compound was found in dichloromethane extract namely: 3-O-methyl-d-glucose (100%) was recorded as the principle constituents. The antibacterial activities of dichloromethane and hexane extracts were measured against three pathogenic bacteria, which are Staphylococcus aureus, Escherichia coli and Streptococcus Pneumonia using diffusion method. Significant dissimilarities in the antibacterial activity of plant extracts were noticed depending on the type of bacterial strain and the concentration of plant extracts.

Fourier transform infrared spectroscopy: unlocking fundamentals and prospects for bacterial strain typing.

The need to identify highly related bacterial strains is ancient in clinical, industrial, or environmental microbiology. Strategies based on different phenotypic and genotypic principles have been used since the early 1930s with variable outcomes and performances, accompanying the evolution of bacterial features' knowledge as well as technologies, instruments, and data analysis tools. Today, more than ever, the implementation of bacterial typing methods that combine a high reliability and accuracy with a rapid, low-cost, and user-friendly performance is highly desirable, especially for clinical microbiology. FT-IR developments for bacterial discrimination at the infra-species level settled on the identification of bacterial groups previously defined by phenotypic or genotypic typing methods. Therefore, this review provides a brief historical overview of main bacterial strain typing methods, and a comprehensive analysis of the fundamentals and applications of Fourier transform infrared spectroscopy, a phenotypic-based method with potential for routine strain typing. The different studies on FT-IR-based strain typing of diverse Gram-negative and Gram-positive bacterial species are discussed in light of genotypic, phenotypic, and biochemical aspects, in order to definitively give this methodology credit to be widely accepted by microbiologists. Importantly, the discriminatory biochemical fingerprints observed on FT-IR spectra have been consistently correlated with sugar-based coating structures that besides reflecting strain variation are also of high relevance for the specificity in pathogen-host interactions. Thus, FT-IR-based bacterial typing might not only be useful for quick and reliable strain typing but also to help understanding the diversity, evolution, and host adaptation factors of key bacterial pathogens or subpopulations.

English

The objective of the present study is to carry out bio-guided phytochemical investigation of Guiera senegalensis and Pseudocedrela kotschyi stem extracts. The two plants are used as toothpicks for oral hygiene. The inhibition test revealed a bacteriostatic effect of hexane extract PK1 of P. kotschyi against Streptococcus mutans ATCC 25175 and Streptococcus salivarius ATCC 20560, two bacterial cariogenic strains, with a decrease in the number of bacterial colonies of 1 Log/control. The aqueous extract GS5 obtained from the stems of G. senegalensis is bactericidal, with total inhibition of S. salivarius ATCC 20560. The antimicrobial effect of the stem extracts from the two plants studied varies according to the plant species and the type of bacterial strain. The phytocompounds 8-Hydroxy-6,7-dimethoxy-3-methylisochroman-4-one, 1-(4-hydroxy-3-methoxyphe-nyl) propane-1,2-dione and (4E, 15E) -Nonadeca-4,15- dien-10-one were isolated, respectively from GS5A extracts of G. senegalensis and PK1 of P. kotschyi by normal column chromatography. Key words: Guiera senegalensis, Pseudocedrela kotschyi, stem extracts, Streptococcus mutans, Streptococcus salivarius, spectroscopic characterization. &nbsp

The antimicrobial spectrum of lysozyme broadened by reductive modification.

In order to broaden the antimicrobial action spectrum of lysozyme against Gram-negative bacteria, different partially reduced lysozyme derivatives were obtained by reducing the R-S-S-R bonds of lysozyme using the reducing agent Na2SO3. The circular dichroism behavior analysis showed that the tertiary structure of the partially reduced lysozyme molecules became more flexible, leading to an obvious increase in the surface hydrophobicity and the tryptophan fluorescence intensity. Zeta potential analysis indicated that the introduction of SO32- led to a decrease in the surface charges of the reduced lysozyme molecules. Compared with the native lysozyme, the reduced lysozymes not only showed a 1 log increase in the antimicrobial activity against Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076 (P <0.05), but also maintained nearly the same antimicrobial activity against Staphylococcus aureus ATCC 29213 (P >0.05). Overall, the reductive modification with the food-friendly, compatible, and safe reducing agent Na2SO3 has broadened the antimicrobial action spectrum of the modified lysozymes against Gram-negative bacteria, with the enhancement extent depending on the reduction degree and the type of bacterial strains. The integrated results suggest that the Na2SO3-reduced lysozyme can be used as a novel safe potential bactericidal additive for food-processing industry.

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.