Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, β-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 μg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17β-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17β-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17β-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.
- MeSH
- chemické látky znečišťující vodu analýza MeSH
- cholestadienoly MeSH
- cholesterol analogy a deriváty MeSH
- estradiol analýza MeSH
- estrogeny analýza MeSH
- estron analýza MeSH
- fytoestrogeny analýza MeSH
- fytosteroly MeSH
- genistein analýza MeSH
- HeLa buňky MeSH
- isoflavony analýza MeSH
- lidé MeSH
- receptory pro estrogeny metabolismus MeSH
- sinice účinky léků metabolismus MeSH
- sitosteroly analýza MeSH
- sladká voda MeSH
- steroly analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- voda MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The present study demonstrates development of a rapid testing protocol based on a small portable luminometer using flash kinetic assessment of bacterial bioluminescence. The laboratory comparisons based on six model organic toxicants and two metals showed significant correlations between responses of freshwater bacteria Photorhabdus luminescens and standard marine bacterial species Vibrio fisheri. While P. luminescens was less sensitive in standard arrangements, the responses of both organisms were comparable in the newly introduced portable luminometer setup. The applicability and reproducibility of the portable luminometer protocol was further demonstrated in the assessment of 43 European wastewater effluents that were simultaneously tested for toxicity and analysed for 150 organic and 20 inorganic contaminants grouped into 13 major chemical classes. Clear association between the toxic responses in both compared bacterial species and the elevated levels of inorganic compounds (toxic metals), chlorophenols and benzotriazole anticorrosives was observed. The new protocol with a portable luminometer provides a fast (30 s) response and may be used as a tool for rapid in situ toxicity evaluation of freshwater environmental samples such as effluents.
Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.
- MeSH
- bakteriální geny * MeSH
- Cronobacter klasifikace genetika MeSH
- DNA bakterií MeSH
- DNA primery MeSH
- DNA řízené RNA-polymerasy genetika MeSH
- fylogeneze MeSH
- kontaminace potravin analýza MeSH
- polymerázová řetězová reakce ekonomika metody MeSH
- polymorfismus délky restrikčních fragmentů * MeSH
- potravinářská mikrobiologie * MeSH
- restrikční endonukleasy typu II metabolismus MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
Strains of Bifidobacterium animalis subsp. lactis are well-known health-promoting probiotics used commercially. B. animalis subsp. lactis has been isolated from different sources, and little is known about animal isolates of this taxon. The aim of this study was to examine the genotypic and phenotypic diversity between B. animalis subsp. lactis strains different animal hosts including Cameroon sheep, Barbary sheep, okapi, mouflon, German shepard and to compare to BB12, food isolates and the collection strain DSM 10140. Ten strains of B. animalis subsp. lactis from different sources were characterised by phenotyping, fingerprinting, and multilocus sequence typing (MLST). Regardless of origin, MLST and phylogenetic analyses revealed a close relationship between strains of B. animalis subsp. lactis with commercial and animal origin with the exception of isolates from ovine cheese, mouflon and German Shepard dog. Moreover, isolates from dog and mouflon showed significant differences in fermentation profiles and peptide mass fingerprints (MALDI-TOF). Results indicated phenotypic and genotypic diversity among strains of B. animalis subsp. lactis.
- MeSH
- Bifidobacterium animalis chemie klasifikace genetika izolace a purifikace fyziologie MeSH
- fenotyp * MeSH
- fylogeneze MeSH
- genetická variace * MeSH
- genotyp * MeSH
- molekulární typizace MeSH
- potravinářská mikrobiologie * MeSH
- savci mikrobiologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- techniky typizace bakterií MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.
- MeSH
- Cronobacter chemie klasifikace genetika izolace a purifikace MeSH
- DNA bakterií genetika MeSH
- DNA fingerprinting MeSH
- enterobakteriální infekce mikrobiologie MeSH
- genetická variace * MeSH
- genotyp MeSH
- lidé MeSH
- multigenová rodina MeSH
- multilokusová sekvenční typizace MeSH
- O-antigeny analýza genetika MeSH
- oligonukleotidové sondy MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- séroskupina MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RATIONALE: The bacterial genus Cronobacter was established quite recently, in 2008. Therefore, its systematic classification is still in progress as well as the risk assessment of Cronobacter strains. The possibility of rapid identification within the biogroup level has an essential epidemiological significance. We examined the potential of mass spectrometry to accomplish this task on species Cronobacter sakazakii comprising eight different biogroups. METHODS: Members of all Cronobacter sakazakii biogroups were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using intact cells. Analyses were performed on a Biflex IV MALDI-TOF mass spectrometer in the range of 2000 to 20 000 Da in linear mode with an accelerated voltage of 19 kV. RESULTS: Optimal conditions for a proper identification of biogroups, such as suitable cultivation media or growth time of bacteria, were investigated. The biomarker patterns characterizing each of the Cronobacter sakazakii biogroups were obtained. The established identification protocol was applied to ten previously non-identified strains and their biogroups were successfully determined. CONCLUSIONS: The presented work is the first report of successful and rapid bacterial biogroup taxonomy classification using MALDI-TOF-MS that could substitute demanding biochemical testing.
- MeSH
- biologické markery analýza chemie MeSH
- Cronobacter sakazakii chemie klasifikace růst a vývoj MeSH
- kultivační média chemie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- shluková analýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- techniky typizace bakterií metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Both immunochemical methods, sandwich ELISA and immunochromatographic detection, for detection of genus Cronobacter, have been developed. Rabbit polyclonal antibodies against dead cells of Cronobacter sakazakii Cb03 (ATCC 29544) were used. In tests of bacteria cultures from microbial collection, the ELISA distinguished between all assessed Cronobacter and Enterobacter strains. The immunochromatographic technique provided false negative results for 20 % of Cronobacter strains, and 40 % false positive results for Enterobacter strains.
Members of the genus Cronobacter are opportunistic pathogens formerly known as Enterobacter sakazakii, which induce severe meningitis and sepsis in neonates and infants, with a high fatality rate. In this work, a simple and rapid immunochromatographic strip test for the detection of this pathogen was developed. Following the shortened bacteria cultivation and isolation of DNA, a specific gene sequence targeting 16S rRNA from Cronobacter spp. was amplified by PCR using 5'-end labelled specific primers. The PCR product, amplicon labelled with digoxigenin on one side and biotin on the other side, was directly added to the immunochromatographic strip test, composed of nitrocellulose membrane with bound antibody against digoxigenin in the test line. The visualization was mediated by colloidal carbon conjugated to neutravidin, and the appearance of grey/black line was indicative of the presence of specific amplicon. Colour intensity of the test line in pathogen-positive assay was visually distinguishable from that of negative sample within 10 min. The visual detection limit of PCR product was 8 ng. The specificity of the developed method was confirmed by standard microbiological techniques. Whole detection procedure with the incorporated immunostrip was applied to analysis of infant formulae samples, contaminated with less than 10 cells of Cronobacter spp. per 10 g. The results from immunochromatographic test indicated the absolute agreement with those from standard microbiological methods. Moreover, the developed procedure considerably reduced the total analysis time to 16 h whereas the reference microbiological method needs 6-7 days.
- MeSH
- bakteriální RNA genetika MeSH
- biosenzitivní techniky metody MeSH
- chromatografie metody MeSH
- Cronobacter sakazakii klasifikace genetika izolace a purifikace patogenita MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- Enterobacteriaceae klasifikace genetika izolace a purifikace patogenita MeSH
- kojenec MeSH
- lidé MeSH
- náhražky mateřského mléka MeSH
- polymerázová řetězová reakce metody MeSH
- potravinářská mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- sekvence nukleotidů MeSH
- techniky typizace bakterií metody MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
