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.
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.
- MeSH
- biofilmy * MeSH
- biologický transport MeSH
- DNA bakterií chemie genetika metabolismus MeSH
- extracelulární prostor mikrobiologie MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- Listeria monocytogenes chemie genetika fyziologie MeSH
- listeriové infekce mikrobiologie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus chemie genetika fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Biofilm matrix forms a dynamic microenvironment facilitating the resilience of the biofilm. Extracellular DNA (eDNA) is one of the matrix components playing a crucial role in the cell adhesion, aggregation and structure stabilization. Its sticky nature and negative charge reduce the penetration of antimicrobials and further enable the biofilm tolerance or even resistance. The resistance can be encoded in the biofilm matrix genetic information. Moreover, the biofilm matrix with eDNA creates an ideal environment for gene transfer. For all above mentioned reasons, eDNA is a substance with a significant potential for the biofilm disruption in food processing, as well as in medicine. The combination of several different techniques successful in biofilm disruption could facilitate even eradication of the biofilm with various pathogens.
- Klíčová slova
- narušení biofilmu,
- MeSH
- bakteriální léková rezistence MeSH
- biofilmy * účinky léků MeSH
- DNA bakterií MeSH
- extracelulární matrix MeSH
- rekombinace genetická MeSH
- Publikační typ
- práce podpořená grantem MeSH
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.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny genetika MeSH
- bakteriální léková rezistence genetika MeSH
- biofilmy * MeSH
- DNA bakterií genetika metabolismus MeSH
- extracelulární prostor genetika MeSH
- lidé MeSH
- Listeria monocytogenes účinky léků genetika růst a vývoj izolace a purifikace MeSH
- mikrobiální testy citlivosti MeSH
- mikrobiální viabilita účinky léků MeSH
- potravinářská mikrobiologie MeSH
- shluková analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
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.
- MeSH
- antibióza * MeSH
- buňky HT-29 MeSH
- Carnobacterium fyziologie MeSH
- endocytóza * MeSH
- epitelové buňky mikrobiologie MeSH
- lidé MeSH
- Listeria monocytogenes patogenita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
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.
- MeSH
- čištění vody MeSH
- dekontaminace * MeSH
- dezinfekce * MeSH
- lidé MeSH
- nádory terapie MeSH
- obaly potravin MeSH
- plazmové plyny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
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.
- Publikační typ
- časopisecké články MeSH
Alimentární infekce způsobené různými potravinovými patogeny v dnešní době obecně představují nezanedbatelnou hrozbu pro veřej - né zdraví. Nicméně využití tradičních a standardizovaných ISO metod pro kontrolu potravin postrádá potřebnou rychlost a spolehlivost pro detekci, identifikaci a kvantifikaci těchto mikroorganismů. Zejména v případě termotolerantních Campylobacter spp., jež jsou v sou - časné době nejčastější příčinnou lidských gastroenteritid bakteriálního původu, je využití těchto metod v praxi obzvláště problematické. Praktickým východiskem výše popsaných nedostatků kultivačních metod je bezesporu zavedení rozličných molekulárně-genetických technik.
Alimentary infections caused by various food-borne pathogens generally pose a threat to public health. The standardised ISO methods usually used for food control sometimes lack swiftness and reliability necessary for detection, identification and quantification of these microorganisms. Especially in the case of thermotolerant Campylobacter spp., which are the most common cause of human bacterial gastroenteritis worldwide, usage of these methods in practice is somehow problematic. Practical solution of abovementioned issues linked with cultivation based methods are indisputably various molecular-genetic techniques
- MeSH
- bakteriální infekce diagnóza MeSH
- Campylobacter * izolace a purifikace MeSH
- kampylobakterové infekce * diagnóza MeSH
- kontaminace potravin * MeSH
- lidé MeSH
- nemoci přenášené potravou mikrobiologie MeSH
- polymerázová řetězová reakce metody využití MeSH
- potravinářská mikrobiologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Biofilmy obklopené extracelulárními polymerními látkami představují jeden z nejčastěji objevujících se fenotypů v přírodě. Biofilmy jsou proto nedílnou součástí přirozených ekosystémů. Mikroorganismy interagují mezi sebou ale i s prostředím, ve kterém žijí. Přítomnost extracelulárních polymerních substancí na površích v kontaktu s jídlem umožňuje kolonizaci potenciálně nebezpečných patogenů nebo mikroorganismů hniloby a usnadňuje kontaminační proces. Hlubší poznání jednotlivých složek matrice může podpořit rozvoj strategií umožňujících účinné rozrušení a odstranění biofilmu. Tento článek je věnován především představení složek matrice a jejich základním funkcím.
Biofilms encased by extracellular polymeric substances are very common phenotype across the nature. Therefore they are inseparable part of natural habitats. Microorganisms interact with each other and also with environment they live in. Presence of extracellular polymeric substances on the surface facilitates colonisation of potentially dangerous pathogens or food spoilage. Better understanding of matrix components can support the development of treatment strategies that successfully disrupt biofilm structure. This article aims to characterise the matrix components and their basic functions.
- MeSH
- bakteriální adheziny biosyntéza chemie MeSH
- bakteriální polysacharidy biosyntéza chemie MeSH
- bakteriální proteiny biosyntéza metabolismus MeSH
- biofilmy * růst a vývoj MeSH
- biopolymery metabolismus škodlivé účinky MeSH
- DNA bakterií metabolismus MeSH
- extracelulární matrix - proteiny metabolismus škodlivé účinky MeSH
- Publikační typ
- práce podpořená grantem MeSH
