Contribution to determination of extracellular DNA origin in the biofilm matrix.

This study is focused on the analysis of extracellular DNA (eDNA) from a biofilm matrix formed by Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. The presence of eDNA in the biofilm of all the studied strains was confirmed by confocal laser scanning microscopy using fluorescent dyes with high affinity to nucleic acid. The protocol for eDNA isolation from the biofilm matrix was established, and subsequent characterization of the eDNA was performed. The purified eDNA obtained from the biofilm matrix of all three microorganisms was compared to the genomic DNA (gDNA) isolated from relevant planktonic grown cells. The process of eDNA isolation consisted of biofilm cultivation, its collection, sonication, membrane filtration, dialysis, lyophilisation, and extraction of DNA separated from the biofilm matrix with cetyltrimethylammonium bromide. An amplified fragment length polymorphism (AFLP) was used for comparing eDNA and gDNA. AFLP profiles showed a significant similarity between eDNA and gDNA at the strain level. The highest similarity, with a profile concordance rate of 94.7% per strain, was observed for S. aureus, L. monocytogenes, and S. enterica exhibited lower profiles similarity (78% and 60%, respectively). The obtained results support the hypothesis that the eDNA of studied bacterial species has its origin in the gDNA.

Quantitative evaluation of biofilm extracellular DNA by fluorescence-based techniques.

The formation of a hardly removable biofilm in food processing and clinical settings calls for a deeper understanding of composition of the matrix that protects the biofilm cells, as the crucial matrix component is extracellular DNA (eDNA), participating in adhesion, aggregation and penetration reduction, yet serving as a horizontal gene transfer reservoir. Therefore, we evaluated eDNA release from the biofilm of two pathogens, Listeria monocytogenes and Staphylococcus aureus, with respect to their origin under different culturing condition. Primarily, the biofilms were observed by confocal laser scanning microscopy (CLSM) under conditions mimicking the food processing environment and human body. The eDNA was quantitatively characterised based on its area by IMARIS. Next, the eDNA content and biofilm formation were quantified by spectrophotometry. Data from both sets of experiments were statistically evaluated. The eDNA release varied between the microorganism, culturing conditions and the origin of strains. Independent of the method used, the clinical strains of S. aureus released more eDNA than the food related strains at 37 °C. eDNA content can be crucial discriminating matrix component between food related and clinical strains. Deeper understanding of the eDNA role in such a phenomenon could facilitate the design of effective strategy for biofilm disruption.

Monitoring of resistance genes in Listeria monocytogenes isolates and their presence in the extracellular DNA of biofilms: a case study from the Czech Republic.

The alarming occurrence of antibiotic resistance genes in food production demands continuous monitoring worldwide. One reservoir of resistance genes is thought to be eDNA. There is currently little available information in Europe about either the extracellular DNA distribution of the bacterium or the spread of resistance genes in L. monocytogenes. Therefore, our aims were to give insight into the Listeria monocytogenes resistance situation in the Czech Republic and assess the presence of resistance genes in their extracellular DNA (eDNA). First, susceptibility tests were performed on 49 isolates of L. monocytogenes with selected antibiotics. Next, we tested DNA of suspected isolates for the presence of resistance genes in both planktonic cells and the eDNA of biofilms. Finally, fluorescent confocal microscopy was used to observe the eDNA pattern of selected isolates under conditions that mimicked the food processing environment and the human body. Susceptibility tests found isolates intermediate resistant to chloramphenicol, tetracycline, and ciprofloxacin as well as isolates resistant to ciprofloxacin. For all suspected isolates, PCR confirmed the presence of the gene lde encoding efflux pump in both types of DNA. When the biofilm was observed using confocal laser scanning microscope, the eDNA distribution patterns varied considerably according to the culture conditions. Furthermore, the food and clinical isolates varied in terms of the amount of eDNA detected. The presence of an efflux pump in both types of DNA suggests that the eDNA might serve as a reservoir of resistance genes. Surprising differences were observed in the eDNA pattern. Our results suggest that the current risk of the spread of L. monocytogenes resistance genes is low in the Czech Republic, but they also indicate the need for continuous long-term monitoring of the situation.

Profiling of Campylobacter jejuni Proteome in Exponential and Stationary Phase of Growth.

Campylobacter jejuni has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO2, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As C. jejuni is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in C. jejuni and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of C. jejuni was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among C. jejuni strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential.

Protective Effect of Carnobacterium spp. against Listeria monocytogenes during Host Cell Invasion Using In vitro HT29 Model.

The pathogenesis of listeriosis results mainly from the ability of Listeria monocytogenes to attach, invade, replicate and survive within various cell types in mammalian tissues. In this work, the effect of two bacteriocin-producing Carnobacterium (C. divergens V41 and C. maltaromaticum V1) and three non-bacteriocinogenic strains: (C. divergens V41C9, C. divergens 2763, and C. maltaromaticum 2762) was investigated on the reduction of L. monocytogenes Scott A plaque-forming during human infection using the HT-29 in vitro model. All Carnobacteria tested resulted in a reduction in the epithelial cell invasion caused by L. monocytogenes Scott A. To understand better the mechanism underlying the level of L. monocytogenes infection inhibition by Carnobacteria, infection assays from various pretreatments of Carnobacteria were assessed. The results revealed the influence of bacteriocin production combined with a passive mechanism of mammalian cell monolayers protection by Carnobacteria. These initial results showing a reduction in L. monocytogenes virulence on epithelial cells by Carnobacteria would be worthwhile analyzing further as a promising probiotic tool for human health.

The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis.

Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.

Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions.

During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

Nonthermal plasma--A tool for decontamination and disinfection.

By definition, the nonthermal plasma (NTP) is partially ionized gas where the energy is stored mostly in the free electrons and the overall temperature remains low. NTP is widely used for many years in various applications such as low-temperature plasma chemistry, removal of gaseous pollutants, in gas-discharge lamps or surface modification. However, during the last ten years, NTP usage expanded to new biological areas of application like plasma microorganisms' inactivation, ready-to-eat food preparation, biofilm degradation or in healthcare, where it seems to be important for the treatment of cancer cells and in the initiation of apoptosis, prion inactivation, prevention of nosocomial infections or in the therapy of infected wounds. These areas are presented and documented in this paper as a review of representative publications.

Detection, identification and quantification of Campylobacter jejuni, coli and lari in food matrices all at once using multiplex qPCR.

BACKGROUND: Thermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR. RESULTS: Species specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low. CONCLUSIONS: Proposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook.

Chemostat-induced uneven division of Bacillus subtilis.

Anomalous forms of Bacillus subtilis A32 produced by prolonged cultivation in a chemostat under nitrogen limitation are described. A change in the cultivation conditions brought about a transformation of these forms to bacillar rods. The transformation was gradual and lasted for several generations.

Effect of methyl tert-butyl ether in standard tests for mutagenicity and environmental toxicity.

Methyl tert-butyl ether (MTBE) is a synthetic compound that is used as a technological solution to problems created by air pollution from vehicle emissions. An important source of MTBE in the environment is leakage from underground storage tanks at gasoline stations or accidents during gasoline transport. The aim of this study was to evaluate the risk of MTBE leakage for the environment using the Microtox (Vibrio fischeri) toxicity test, Lactuca sativa seed germination test, and Ames bacterial mutagenicity test with Salmonella typhimurium his(-) strains TA98, TA100, YG1041, and YG1042, using both standard plate and preincubation protocols. The result of Microtox expressed as EC(50) was 33 mg (MTBE)/L. The effect of all tested MTBE concentration (0.05, 0.50, and 1.00% v/v) on Lactuca sativa roots elongation was negative and proved its toxicity. The highest tested concentration of MTBE that could be tested in Ames test was 3 mg (MTBE)/plate, because of cytotoxicity. No mutagenic response was observed at this or lower concentrations in any of the four strains used.

External factors involved in the regulation of synthesis of an extracellular proteinase in Bacillus megaterium: effect of temperature.

We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35-38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24-28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26-34° C) when the rate of temperature decrease was 0.2-0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32-34° C. The optimum temperature decrease was 0.8° C/h.