Zvyšující se výskyt antibiotických rezistencí patří k závažným problémům 21.století. Výskyt bakteriálních kmenů rezistentních k antibiotikům následně zužuje spektrum vhodných antibiotik použitelných pro léčbu i běžných bakteriálních infekcí nebo pro prevenci jejich výskytu, např. v chirurgii. Čistírny odpadních vod, nemocnice, ale i potravinový řetězec patří k ohniskům, kde nejčastěji dochází ke vzniku či šíření nových i stávajících kmenů bakterií rezistentních k antibiotikům a genů rezistence k antibiotikům. Ke stanovení antibiotických rezistencí se v laboratořích standardně používají fenotypové kultivační metody, které jsou však náročné na čas i práci a částečně i přesnou interpretaci výsledků. Z tohoto důvodu jsou hledány rychlejší alternativní metody detekce bakterií rezistentních k antibiotikům nebo přímo genů rezistence k antibiotikům. Příkladem alternativní metody detekce bakterií rezistentních k antibiotikům je například použití fenotypové metody využívající hmotnostní spektrometrie s laserovou desorpcí a ionizací za účasti matrice s průletovým analyzátorem pro stanovení producentů beta-laktamas. Zrychlení a zároveň větší přesnost detekce poskytují genotypové metody. Pomocí polymerasové řetězové reakce lze přímo detekovat a kvantifikovat geny rezistence k antibiotikům. Pro další zrychlení a vyšší specifitu detekce amplikonů z PCR lze použít mikročipy. Metody masivního paralelního sekvenování poskytují ucelenou informaci o rezistomu daného prostředí. Umožňují sekvenovat DNA amplikony či jednotlivé molekuly DNA pro detekci determinant antibiotické rezistence. Metody masivního paralelního sekvenování mají potenciál nahradit konvenční charakterizaci patogenů a umožňují detekci všech mikroorganismů ve vzorku (včetně obtížně kultivovatelných či nekultivovatelných mikroorganismů).
The increasing occurrence of antibiotic resistance is one of the major problems of the 21st century. The occurrence of bacterial strains resistant to antibiotics subsequently narrows the spectrum of suitable antibiotics usable for the treatment of common bacterial infections or for the prevention of their occurrence, e.g., in surgery. Wastewater treatment plants, hospitals, and also the food chain belong to the hotspots, where the emergence and spread of new or existing strains of antibiotic resistant bacteria and antibiotic resistance genes occur most frequently. Phenotypic culture methods are routinely used in laboratories to determine antibiotic resistance, but they are laborious and time-consuming and the interpretation of exact results is also difficult. For this reason, faster alternatives for the detection of antibiotic resistant bacteria or even antibiotic resistance genes are sought. Such an example of an alternative method for the detection of antibiotic resistant bacteria is the use of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry phenotypic method to identify the beta-lactamase producers. Genotype methods provide faster analysis and, at the same time, more accurate detection. Antibiotic resistance genes can be directly detected and quantified by polymerase chain reaction. Microarrays can be used to further speed up and increase the specificity of PCR amplicons detection. Massive parallel methods provide comprehensive information on the resistoma of the specific environment. They facilitate sequencing of individual DNA molecules or amplicons to detect determinants of antibiotic resistance. Massive parallel methods have the potential to replace conventional pathogen characterization and allow the detection of all microorganisms in a sample (including difficult-to-cultivate or noncultivable microorganisms).
- MeSH
- antibiotická rezistence * genetika MeSH
- mikrobiální testy citlivosti metody MeSH
- mikrobiologické techniky * klasifikace metody MeSH
- polymerázová řetězová reakce metody MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- přehledy MeSH
In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.
- MeSH
- acetylcystein farmakologie MeSH
- antibakteriální látky farmakologie MeSH
- antiinfekční látky farmakologie MeSH
- benzofurany farmakologie MeSH
- biofilmy účinky léků MeSH
- buněčné linie MeSH
- Escherichia coli účinky léků MeSH
- glykolipidy farmakologie MeSH
- kontaminace potravin prevence a kontrola MeSH
- lidé MeSH
- Listeria monocytogenes účinky léků MeSH
- mikrobiální testy citlivosti MeSH
- nemoci přenášené potravou farmakoterapie mikrobiologie MeSH
- potravinářská mikrobiologie metody MeSH
- Salmonella enterica účinky léků MeSH
- Staphylococcus aureus účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Although some metallic nanoparticles (NPs) are commonly used in the food processing plants as nanomaterials for food packaging, or as coatings on the food handling equipment, little is known about antimicrobial properties of palladium (PdNPs) and platinum (PtNPs) nanoparticles and their potential use in the food industry. In this study, common food-borne pathogens Salmonella enterica Infantis, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus were tested. Both NPs reduced viable cells with the log10 CFU reduction of 0.3-2.4 (PdNPs) and 0.8-2.0 (PtNPs), average inhibitory rates of 55.2-99% for PdNPs and of 83.8-99% for PtNPs. However, both NPs seemed to be less effective for biofilm formation and its reduction. The most effective concentrations were evaluated to be 22.25-44.5 mg/L for PdNPs and 50.5-101 mg/L for PtNPs. Furthermore, the interactions of tested NPs with bacterial cell were visualized by transmission electron microscopy (TEM). TEM visualization confirmed that NPs entered bacteria and caused direct damage of the cell walls, which resulted in bacterial disruption. The in vitro cytotoxicity of individual NPs was determined in primary human renal tubular epithelial cells (HRTECs), human keratinocytes (HaCat), human dermal fibroblasts (HDFs), human epithelial kidney cells (HEK 293), and primary human coronary artery endothelial cells (HCAECs). Due to their antimicrobial properties on bacterial cells and no acute cytotoxicity, both types of NPs could potentially fight food-borne pathogens.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- Bacteria klasifikace účinky léků růst a vývoj MeSH
- fibroblasty cytologie účinky léků MeSH
- kovové nanočástice aplikace a dávkování chemie MeSH
- kultivované buňky MeSH
- ledviny cytologie účinky léků MeSH
- lidé MeSH
- nemoci přenášené potravou prevence a kontrola MeSH
- palladium chemie MeSH
- platina chemie MeSH
- potravinářská mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The antimicrobial activity of gold and silver nanoparticles (AuNPs, AgNPs), chitosan (CS) and their combinations was established by determining the minimum inhibitory concentration for planktonic (MICPC80) and biofilm growth (MICBC80), for biofilm formation (MICBF80), metabolic activity (MICBM80) and reduction (MICBR80), and for the metabolic activity of preformed biofilm (MICMPB80). Biofilms were quantified in microtitre plates by crystal violet staining and metabolic activity was evaluated by the MTT assay. Chitosan effectively suppressed biofilm formation (0.31-5 mg ml-1) in all the tested strains, except Salmonella enterica Infantis (0.16-2.5 mg ml-1) where CS and its combination with AgNPs induced biofilm formation. Nanoparticles inhibited biofilm growth only when the highest concentrations were used. Even though AuNPs, AgNPs and CS were not able to remove biofilm mass, they reduced its metabolic activity by at least 80%. The combinations of nanoparticles with CS did not show any significant positive synergistic effect on the tested target properties.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- biofilmy účinky léků růst a vývoj MeSH
- chitosan chemie farmakologie MeSH
- gramnegativní bakterie účinky léků MeSH
- grampozitivní bakterie účinky léků MeSH
- kovové nanočástice chemie MeSH
- mikrobiální testy citlivosti MeSH
- potravinářská mikrobiologie MeSH
- stříbro chemie farmakologie MeSH
- synergismus léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zlato chemie farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Staphylococcus aureus is a major food-borne pathogen due to the production of enterotoxin and is particularly prevalent in contaminated milk and dairy products. The lactic acid bacteria (LAB) are widely used as biocontrol agents in fermented foods which can inhibit pathogenic flora. In our work, we investigated the influence of three strains of LAB (Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus durans) on the relative expression of three enterotoxin genes (sea, sec, sell) and eight virulence and/or regulatory genes (sarA, saeS, codY, srrA, rot, hld/RNAIII, agrA/RNAII, sigB) in two S. aureus strains (MW2 and Sa1612) in TSB and reduced-fat milk (1.5 %) at 30 °C over a 24-h period. The tested LAB and S. aureus strains proved to be mutually non-competitive or only slightly competitive during co-cultivation. In addition, under the above-mentioned conditions, differential gene expression between the S. aureus MW2 and Sa1612 strains was well documented. S. aureus growth was changed in mixed culture with LAB; however, its effect on the repression of sea and sec expression correlated with production of these virulence factors. In comparison, the presence of LAB strains generally inhibited the expression of sec, sell, sarA, seaS, agrA/RNAII and hld/RNAIII genes. The effect of LAB strains presence on the expression of sea, codY, srrA, rot and sigB genes was medium, time, LAB and S. aureus strain specific. SEA and SEC production was significantly reduced in milk compared to TSB in pure culture. After the 24-h cultivation, S. aureus MW2 and Sa1612 SEC production was 187 and 331 times lower in milk compared to TSB, respectively (0.07 and 0.39 ng/mL in milk, versus 13.1 and 129.2 ng/mL in TSB, respectively). At the same time S. aureus MW2 and Sa1612 SEA production was 77 and 68 times lower in milk compared to TSB, respectively (0.99 and 0.17 ng/mL in milk, versus 76.4 and 11.5 ng/mL in TSB, respectively). This study has revealed new insights into the interaction between S. aureus and LAB (L. plantarum, S. thermophilus, E. durans) on the level of the expression and/or production of S. aureus enterotoxins, regulatory and virulence genes in different media, including milk. This study provides data which may improve the quality of food production.
- MeSH
- Enterococcus fyziologie MeSH
- enterotoxiny biosyntéza genetika MeSH
- faktory virulence genetika MeSH
- kokultivační techniky MeSH
- kultivační média MeSH
- Lactobacillus plantarum fyziologie MeSH
- mikrobiální interakce MeSH
- mléko * MeSH
- potravinářská mikrobiologie MeSH
- regulační geny MeSH
- Staphylococcus aureus genetika růst a vývoj metabolismus patogenita MeSH
- Streptococcus thermophilus fyziologie MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
