In Vitro Antibacterial Activity of Phlorotannins from Edible Brown Algae, <Emphasis Type="Italic">Eisenia bicyclis</Emphasis> Against Streptomycin-Resistant <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

Listeria monocytogenes (LM) is an important food borne pathogen responsible for listeriosis. Further, LM is an etiological agent associated with life threatening conditions like meningitis and encephalitis. Biofilm forming and drug resistant LM may potentially become difficult to treat infections and hence effective controlling measures are required to prevent LM infections. In view of this, the present study evaluated an anti-listerial potential of edible brown seaweed, Eisenia bicyclis, by disc diffusion and micro-dilution methods. The results of the present study suggested that the anti-listerial activity of various phlorotannins isolated form E. bicyclis were in the range of 16–256 µg/ml. Among the phlorotannins isolated, fucofuroeckol-A (FAA) exhibited the highest anti-listerial potential (MIC range 16–32 µg/ml) against LM strains tested. Further, in checker board synergy assays, FFA-streptomycin combination exhibited significant synergy (fractional inhibitory concentration index, ∑FIC < 0.5) against aminoglycoside resistant clinical strains of LM. The results of the present study suggested the potential use of edible seaweed E. bicyclis as a source of natural phlorotannins to control food borne pathogenic infections.     

Survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Wilted and Additive-Treated Grass Silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10⁶–10⁷ cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in food-borne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8·10⁵/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25°C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10² and 10⁶ cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

<Emphasis Type="Italic">Listeria </Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> internalins bind to the human intestinal mucin MUC2

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.     

Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

Adaptation and cross-adaptation of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Salmonella enterica</Emphasis> to poultry decontaminants

Information on the potential for acquired reduced susceptibility of bacteria to poultry decontaminants occurring is lacking. Minimal Inhibitory Concentrations (MICs) were established for assessing the initial susceptibility and the adaptative and cross-adaptative responses of four bacterial strains (Listeria monocytogenes serovar l/2a, L. monocytogenes serovar 4b, Salmonella enterica serotype Typhimurium, and S. enterica serotype Enteritidis) to four poultry decontaminants (trisodium phosphate, acidified sodium chlorite -ASC-, citric acid, and peroxyacetic acid). The initial susceptibility was observed to differ among species (all decontaminants) and between Salmonella strains (ASC). These inter- and intra-specific variations highlight (1) the need for strict monitoring of decontaminant concentrations to inactivate all target pathogens of concern, and (2) the importance of selecting adequate test strains in decontamination studies. MICs of ASC (0.17±0.02 to 0.21±0.02 mg/ml) were higher than the U.S. authorized concentration when applied as a pre-chiller or chiller solution (0.05 to 0.15 mg/ml). Progressively increasing decontaminant concentrations resulted in reduced susceptibility of strains. The highest increase in MIC was 1.88 to 2.71-fold (ASC). All decontaminants were shown to cause cross-adaptation of strains between both related and unrelated compounds, the highest increase in MIC being 1.82-fold (ASC). Our results suggest that the in-use concentrations of ASC could, in certain conditions, be ineffective against Listeria and Salmonella strains. The adaptative and cross-adaptative responses of strains tested to poultry decontaminants are of minor concern. However, the observations being presented here are based on in vitro studies, and further research into practical applications are needed in order to confirm these findings.     

Biofilm formation by different serological variants of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in association with <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

Ability of Listeria monocytogenes serological variants belonging to phylogenetic lines I and II to form biofilms on glass, both in monoculture and in a consortium with Bacillus pumilus, was shown. After 72 h, both L. monocytogenes serological variants formed mature biofilms both in monoculture and in association with bacilli. The presence of B. pumilus in Listeria biofilms resulted in alteration of L. monocytogenes colony morphology, decrease in their enzymatic activity and aghesive capacity, enhanced virulence and hemolytic activity, and cell motility observed at 37°С. Importantly, all of these modifications of the biological characteristics were of a phenotypic nature and were restored when joint incubation of bacteria was terminated.     

Factors affecting survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in soil samples

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.     

Identification of genes involved in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm formation by <Emphasis Type="Italic">mariner</Emphasis>-based transposon mutagenesis

Listeria monocytogenes is a ubiquitous food-borne pathogen, whose distribution and survival in food-processing environments are associated with the ability to form biofilms. The process of biofilm formation is complex and its molecular mechanism is relatively poorly understood in L. monocytogenes. To better understand the genetics of this process, a mariner-based transposon mutagenesis strategy was used to identify genes involved in biofilm formation of L. monocytogenes. A library of 6,500 mutant colonies was screened for reduced biofilm formation using a microtiter plate biofilm assay. Forty biofilm-deficient mutants of L. monocytogenes were identified based on DNA sequences of the transposon-flanking regions and Southern hybridization with a transposon-based probe. The insertions harbored by these mutants led to the identification of 24 distinct loci, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation in L. monocytogenes. Genetic complementation confirmed the importance of lmo1386, a gene encoding a putative DNA translocase, for biofilm formation. Molecular analyses of mutants indicated that the majority of the 24 identified genes are related to flagella motility, gene regulation, and cell surface structures.     

Subtyping of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> isolates by <Emphasis Type="Italic">actA</Emphasis> gene sequencing,PCR-fingerprinting,and cell-invasion assay

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Linearized kinetic model of<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> biofilm growth

In this study the dynamics of biofilm formation on aluminum has been investigated. The process of cell growth has been observed using fluorescence microscopy. It has been confirmed that the process of biofilm formation can be represented as a sum of two separate processes: cell adhesion and colony proliferation. The derived set of equations describes kinetics of surface population growth and characteristic times for adsorption and combined growth processes, including characteristic time for the nutrient supply depletion. All equations contain variables based on the fundamental characteristics of bacterial population and can be easily determined from the experimental data or estimated theoretically. The developed theoretical model allows obtaining realistic values for population growth time and characteristic time for nutrient limitation occurrence during the biofilm development. Resulting equations qualitatively describe the biofilm formation process, and allow predicting microbial kinetics in the batch reactor system and determining critical values of the process parameters.     

Reversion of nonculturable forms of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> under the influence of exometabolites of <Emphasis Type="Italic">Aranicola</Emphasis> spp.

It was established that the exometabolites of Aranicola spp., isolated from beef, promoted the reversion of nonculturable forms of Listeria monocytogenes, isolated from the same product, into the proliferative state, influencing the expression of their biological properties, which was expressed by a change in the morphology and enzymatic properties of the colony, and also by an increase the hemolytic activity and a decrease in adhesiveness. It was noted that all changes in the biological properties of L. monocytogenes were phenotypic. Passage in trypticase-soy broth with further seeding of Listeria on yeast agar after the third passage led to a gradual change in the biological properties, until their complete recovery after the fifth passage on the sixth day of the study.     

Oleanolic acid and ursolic acid affect peptidoglycan metabolism in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

The plant pentacyclic triterpenoids, oleanolic and ursolic acids, inhibit the growth and survival of many bacteria, particularly Gram-positive species, including pathogenic ones. The effect of these compounds on the facultative human pathogen Listeria monocytogenes was examined. Both acids affected cell morphology and enhanced autolysis of the bacterial cells. Autolysis of isolated cell walls was inhibited by oleanolic acid, but the inhibitory activity of ursolic acid was less pronounced. Both compounds inhibited peptidoglycan turnover and quantitatively affected the profile of muropeptides obtained after digestion of peptidoglycan with mutanolysin. These results suggest that peptidoglycan metabolism is a cellular target of oleanolic and ursolic acids.     

Rapid and Sensitive Detection of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> by Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) was designed for detection of Listeria monocytogenes, which is an important food-borne kind of pathogenic bacteria causing human and animal disease. The primers set for the hlyA gene consist of six primers targeting eight regions on specific gene. The LAMP assay could be performed within 40 min at 65°C in a water bath. Amplification products were visualized by calcein and manganous ion and agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 2.0 CFU per reaction. The LAMP assay was 100-fold higher sensitive than that of the conventional PCR assay. Taking this way, 60 chicken samples were investigated for L. monocytogenes. The accuracy of LAMP was shown to be 100% when compared to the “gold standard” culture-biotechnical, while the PCR assay failed to detect L. monocytogenes in two of the positive samples. It is shown that LAMP assay can be used as a sensitive, rapid, and simple detection tool for the detection of L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.