Components of cyanobacterial water blooms were quantified in aerosols above agitated water surfaces of five freshwater bodies. The thoracic and respirable aerosol fraction (0.1-10 μm) was sampled using a high-volume sampler. Cyanotoxins microcystins were detected by LC-MS/MS at levels 0.3-13.5 ng/mL (water) and < 35-415 fg/m3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10-119 EU/mL (water) and 0.13-0.64 EU/m3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 104-105 cells eq./mL (water) and 101-103 cells eq./m3 (aerosol). Lipopolysaccharides isolated from bloom samples induced IL-6 and IL-8 cytokine release in human bronchial epithelial cells Beas-2B, while extracted cyanobacterial metabolites induced both pro-inflammatory and cytotoxic effects. Bloom components detected in aerosols and their bioactivities observed in upper respiratory airway epithelial cells together indicate that aerosols formed during cyanobacterial water blooms could induce respiratory irritation and inflammatory injuries, and thus present an inhalation health risk.
- MeSH
- aerosoly MeSH
- chromatografie kapalinová MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- mikrocystiny toxicita MeSH
- sinice * metabolismus MeSH
- sladká voda analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- toxiny kmene Cyanobacteria * MeSH
- voda MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).
- MeSH
- bakteriální proteiny izolace a purifikace toxicita MeSH
- cytotoxiny izolace a purifikace toxicita MeSH
- detergenty chemie MeSH
- erytrocyty účinky léků MeSH
- Escherichia coli metabolismus MeSH
- hemolýza MeSH
- hemolyziny izolace a purifikace toxicita MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- močovina chemie MeSH
- nádorové buněčné linie MeSH
- oktoxynol chemie MeSH
- ovce MeSH
- THP-1 buňky MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Colistin is the last hope to treat extensively drug resistance (XDR) Acinetobacter baumannii (A. baumannii) infections, but resistance to colistin is currently reported in clinical centers all over the world. Here, we studied two colistin-resistant A. baumannii isolates with a difference in minimum inhibitory concentrations (MICs) that were isolated from a single burn patient during treatment in the hospitalization period. The international clonal (IC) lineage, multilocus sequence typing (MLST), and multiple loci variable number tandem repeat (VNTR) analysis (MLVA) typing were used to characterize the relatedness of A. baumannii isolates. Lipopolysaccharides (LPS) and PmrAB system analysis by PCR sequencing, polyacrylamide gel electrophoresis (PAGE), and real-time PCR were performed to determine the intactness and probable modifications of the LPS as the main resistance mechanisms to colistin. A combination of PCR, sequencing, and restriction fragment length polymorphism (RFLP) was used for A. baumannii resistance islands (AbaR) mapping as resistance-determinant reservoirs. Two isolates were identical at all MLST and VNTR marker loci that indicated the isolates were the same strain. In comparison to colistin-heteroresistant A. baumannii strain TEH267 (MIC = 1.5 mg/L), colistin-resistant A. baumannii strain TEH273 (MIC ≥256 mg/L) acquired two genomic regions including Tn6018-topA sequence and topA sequence-3' CS in its AbaR structure containing ispA and cadA genes which, it would appear, could be associated with eightfold increase in colistin MIC. Both isolates had new variants of AbaR-like structures which could be derivatives of the typical AbaR3. According to the results of this study, AbaRs could be associated with an increase in MIC to colistin.
- MeSH
- Acinetobacter baumannii klasifikace účinky léků genetika izolace a purifikace MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny MeSH
- bakteriální proteiny genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- genotyp MeSH
- infekce bakteriemi rodu Acinetobacter mikrobiologie MeSH
- kolistin farmakologie MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- mikrobiální testy citlivosti MeSH
- minisatelitní repetice MeSH
- multilokusová sekvenční typizace MeSH
- polymerázová řetězová reakce MeSH
- popálení komplikace MeSH
- transkripční faktory genetika MeSH
- transpozibilní elementy DNA * 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.
Similar to lipopolysaccharide (LPS), a product of Gram-negative bacteria, the signal macromolecules of Gram-positive bacteria lipoteichoic acid (LTA) and peptidoglycan (PGN) possess multiple biological activities. They may be a source of misinterpretation of experimental findings. We have found that not only LPS but also LTA and PGN can be detected by the Limulus amebocyte lysate (LAL) assay. All of them stimulate the high output in vitro nitric oxide (NO) production of in rat peritoneal cells. The onset of the NO enhancement was observed with 25-100pg/ml of LPS and 25-100ng/ml of PGN and LTA. Polymyxin B (PMX), if applied at concentration 10,000-fold higher than that of LPS, can completely inhibit the NO and LAL binding responses of LPS. The NO-stimulatory and LAL-binding properties of LTA and PGN are not eliminated by PMX. Handling of LPS contamination with PMX may be associated with serious problems because it possesses intrinsic biological activity and becomes cytotoxic at concentration >25μg/ml. The present findings suggest a convenient alternative avoiding these issues. As monitored by the NO and LAL assays, even high amounts of LPS as well as PGN and LTA can be removed by molecular mass cutoff microfiltration. All types of the filters (3kDa to 100kDa) are equally effective. It is suggested that the microfiltration procedure may be considered as a preferable, general and easy method of sample decontamination.
- MeSH
- Bacillus subtilis chemie izolace a purifikace MeSH
- buněčné linie MeSH
- centrifugace MeSH
- Escherichia coli chemie izolace a purifikace MeSH
- filtrace * MeSH
- krysa rodu rattus MeSH
- kyseliny teichoové analýza MeSH
- Limulus test * MeSH
- lipopolysacharidy analýza MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oxid dusnatý biosyntéza metabolismus MeSH
- peptidoglykan analýza MeSH
- potkani Wistar MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- aktivace komplementu genetika imunologie MeSH
- antigeny povrchové analýza imunologie MeSH
- infekce vyvolané Escherichia coli diagnóza imunologie moč MeSH
- interpretace statistických dat MeSH
- lektiny analýza krev MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- sérum imunologie mikrobiologie MeSH
- Check Tag
- lidé MeSH
- MeSH
- bakteriocinové plazmidy analýza MeSH
- finanční podpora výzkumu jako téma MeSH
- kur domácí mikrobiologie MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- membránové proteiny analýza MeSH
- Salmonella enteritidis genetika izolace a purifikace MeSH
- Salmonella typhimurium genetika izolace a purifikace MeSH
- vejce mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
