BACKGROUND: This study aimed to determine the differences in the cervical load and prevalence of Lactobacillus crispatus DNA, Lactobacillus iners DNA, Gardnerella vaginalis DNA, Sneathia sanguinegens DNA, and Ureaplasma species DNA between pregnant women with preterm prelabor rupture of membranes (PPROM) with and without a history of cervical excisional treatment. We also assessed the changes in the cervical load and prevalence of L. crispatus DNA, L. iners DNA, G. vaginalis DNA, S. sanguinegens DNA, and U. spp DNA. according to the cone length. METHODS: This retrospective study included 132 women with singleton pregnancies complicated by PPROM. For all women, information about the cervical loads of bacterial DNA corresponding to L. crispatus, L. iners, G. vaginalis, S. sanguinegens, and U. spp., which was assessed using PCR, was available. RESULTS: Women with a history of cervical excisional treatment had a higher cervical load of L. iners DNA (4.4 × 106 copies DNA/mL vs. 3.5 × 105 copies DNA/mL, p = .04) and a higher load and prevalence of U. spp. DNA (1.1 × 105 copies DNA/mL vs. 9.6 × 104 copies DNA/mL, p = .03; 2.7% vs. 0.5%, p = .04) than those without a history of cervical excisional treatment. In the subset of women with a history of cervical excisional treatment, those with a cone length 18 mm and more had a lower relative abundance of L. crispatus DNA (6% vs. 89%, p = .02), a higher load and relative abundance of L. iners DNA (1.1 × 107 copies DNA/mL vs. 8.2 × 105 copies DNA/mL, p = .04; 91% vs. 35%, p = .04), and higher loads of G. vaginalis DNA (7.6 × 104 copies DNA/mL vs. 3.2 × 102 copies DNA/mL, p = .02) than those with cone length < 18 mm. CONCLUSIONS: A history of cervical excisional treatment was associated with alterations in the cervical microbiota composition in pregnant women with PPROM.
- MeSH
- cervix uteri * mikrobiologie chirurgie MeSH
- DNA bakterií analýza izolace a purifikace MeSH
- dospělí MeSH
- Gardnerella vaginalis izolace a purifikace genetika MeSH
- Lactobacillus izolace a purifikace genetika MeSH
- lidé MeSH
- mikrobiota MeSH
- předčasný odtok plodové vody * mikrobiologie epidemiologie MeSH
- retrospektivní studie MeSH
- těhotenství MeSH
- Ureaplasma izolace a purifikace MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: The aim of this study is to evaluate the diagnostic ability of multiplex real-time polymerase chain reaction (PCR) in very preterm infants assessed for risk of early onset neonatal sepsis (EOS). METHODS: Prospective observational cohort study. Blood samples of preterm neonates ≤32 weeks of gestation were evaluated by commercial multiplex real-time PCR within 2 h after delivery. The definition of EOS was based on positive blood culture and clinical signs of infection or negative blood culture, clinical signs of infection and abnormal neonatal blood count and serum biomarkers. RESULTS: Among 82 subjects analyzed in the study, 15 had clinical or confirmed EOS. PCR was positive in four of these infants (including the only one with a positive blood culture), as well as in 15 of the 67 infants without sepsis (sensitivity 27%, specificity 78%). Out of 19 PCR positive subjects, Escherichia coli was detected in 12 infants (63%). Statistically significant association was found between vaginal E. coli colonization of the mother and E. coli PCR positivity of the neonate (p=0.001). No relationship was found between neonatal E. coli swab results and assessment findings of bacterial DNA in neonatal blood stream. CONCLUSIONS: Multiplex real-time PCR had insufficient diagnostic capability for EOS in high risk very preterm infants. The study revealed no significant association between PCR results and the diagnosis of clinical EOS. Correlation between maternal vaginal swab results and positive PCR in the newborn needs further investigation to fully understand the role of bacterial DNA analysis in preterm infants.
- MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- infekce vyvolané Escherichia coli diagnóza MeSH
- kohortové studie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- novorozenec nedonošený * MeSH
- novorozenec MeSH
- novorozenecká sepse diagnóza MeSH
- těhotenství MeSH
- vagina mikrobiologie MeSH
- vertikální přenos infekce MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- pozorovací studie MeSH
The Gram-negative, obligate intracellular tick-transmitted pathogen Anaplasma phagocytophilum can cause acute febrile diseases in humans and domestic animals. The expansion of the tick Ixodes ricinus (Linnaeus, 1758) in northern Europe due to climate change is of serious concern for animal and human health. The aim of the present study was to investigate the impact of A. phagocytophilum infection in moose Alces alces (Linnaeus) calves by evaluating the carcass weights of infected and non-infected animals and examining animal tissues samples for co-infections with either species of Babesia Starcovici, 1893 or bacteria of the genus Bartonella. The carcasses of 68 free-ranging moose calves were weighed by hunters during the hunting seasons from 2014 to 2017 in two regions in southern Norway and spleen samples were collected. Anaplasma phagocytophilum was detected in moose sampled from locations infected with ticks with a prevalence of 82% (n = 46). The carcass weights of A. phagocytophilum-infected calves (n = 46) and non-infected (n = 22) calves were compared. Although the average weight of infected calves (45.6 kg) was lower than that of non-infected calves (46.5 kg), the difference was not statistically significant. Three different variants of the bacterium 16S rRNA gene were identified. The average weight of animals infected with variant I was 49.9 kg, whereas that of animals infected with variant III was 42.0 kg, but the difference was not statistically significant (p = 0.077). Co-infections of A. phagocytophilum with Bartonella spp. or with Babesia spp. were found in 20 and two calves, respectively. A triple infection was found in two calves. Sequence analysis of the 18S rRNA gene of Babesia-positive samples revealed the presence of Babesia cf. odocoilei (Emerson et Wright, 1970). Strains of Bartonella closely related to Bartonella bovis (Bermond, Boulouis, Heller, Laere, Monteil, Chomel, Sander, Dehio et Piemont, 2002) were identified based on phylogenetic analysis of the gltA and rpoB genes. The loss of body mass in moose calves in the tick-infected site was probably influenced by multiple factors.
- MeSH
- Anaplasma phagocytophilum * klasifikace genetika izolace a purifikace MeSH
- Babesia genetika MeSH
- Bartonella genetika MeSH
- DNA bakterií chemie genetika izolace a purifikace MeSH
- ehrlichióza komplikace epidemiologie patologie veterinární MeSH
- fylogeneze MeSH
- oligonukleotidy chemie MeSH
- polymerázová řetězová reakce veterinární MeSH
- sekvence nukleotidů MeSH
- slezina mikrobiologie patologie MeSH
- tělesná hmotnost MeSH
- vysoká zvěř * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Norsko MeSH
Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.
INTRODUCTION: The aim of this study is to analyse the impact of vaccination of infants with pneumococcal conjugate vaccine (PCV) on the incidence of invasive pneumococcal disease (IPD) in children under 5 years of age in the Czech Republic. MATERIAL AND METHODS: The present study includes all IPD cases reported in children aged 0-4 years within the surveillance program in 2007-2017. The impact of PCV is analysed for five categories of IPD: cases caused by all serotypes, cases caused by PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F), cases caused by three additional PCV10 serotypes (1, 5, and 7F), cases caused by three additional PCV13 serotypes (3, 6A, and 19A), and cases caused by non-PCV serotypes. To assess the impact of PCV, the study period was divided into the pre-vaccination period 2007-2008 and post-vaccination period 2009-2017, which was divided into three three-year parts: 2009-2011, 2012-2014, and 2015-2017. Analysis of differences between periods was based on the Poisson regression model where the population numbers were handled as an offset. RESULTS: The annual incidence of IPD in children under 5 years of age caused by all serotypes has had a downward trend since 2007: it dropped from 8.52/100 000 in 2007 to 2.67/100 000 in 2017, with slight increases in 2010 and 2013. All three post-vaccination periods show significantly lower (p<0.001) incidences in comparison to the pre-vaccination period, but they do not statistically significantly differ from each other. CONCLUSIONS: IPD surveillance data in the Czech Republic show that after the introduction of PCV vaccination of infants, there has been a significant decrease in the IPD incidence of children under 5 years of age. Continued IPD surveillance is essential to monitor for possible post-vaccination serotype replacement.
- MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- incidence MeSH
- kohortové studie MeSH
- kojenec MeSH
- lidé MeSH
- novorozenec MeSH
- ochrana veřejného zdraví metody MeSH
- pneumokokové infekce epidemiologie prevence a kontrola MeSH
- pneumokokové vakcíny terapeutické užití MeSH
- předškolní dítě MeSH
- séroskupina MeSH
- Streptococcus pneumoniae genetika imunologie MeSH
- vakcinace metody MeSH
- vakcíny konjugované terapeutické užití MeSH
- výsledek terapie MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
The anoxygenic phototrophic bacteria (APB) are an active component of aquatic microbial communities. While DNA-based studies have delivered a detailed picture of APB diversity, they cannot provide any information on the activity of individual species. Therefore, we focused on the expression of a photosynthetic gene by APB communities in two freshwater lakes (Cep lake and the Římov Reservoir) in the Czech Republic. First, we analyzed expression levels of pufM during the diel cycle using RT-qPCR. The transcription underwent a strong diel cycle and was inhibited during the day in both lakes. Then, we compared DNA- (total) and RNA-based (active) community composition by sequencing pufM amplicon libraries. We observed large differences in expression activity among different APB phylogroups. While the total APB community in the Římov Reservoir was dominated by Betaproteobacteria, Alphaproteobacteria prevailed in the active library. A different situation was encountered in the oligotrophic lake Cep where Betaproteobacteria (order Burkholderiales) dominated both the DNA and RNA libraries. Interestingly, in Cep lake we found smaller amounts of highly active uncultured phototrophic Chloroflexi, as well as phototrophic Gemmatimonadetes. Despite the large diversity of APB communities, light repression of pufM expression seems to be a common feature of all aerobic APB present in the studied lakes.
- MeSH
- Alphaproteobacteria izolace a purifikace fyziologie účinky záření MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Betaproteobacteria izolace a purifikace fyziologie účinky záření MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- fotoperioda * MeSH
- fotosyntetické reakční centrum - proteinové komplexy genetika metabolismus MeSH
- fototrofní procesy genetika účinky záření MeSH
- fylogeneze MeSH
- jezera mikrobiologie MeSH
- mikrobiota fyziologie účinky záření MeSH
- regulace genové exprese u bakterií fyziologie účinky záření MeSH
- světlo škodlivé účinky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Nitrogen fixation and assimilation processes are vital to the functioning of any ecosystem. Nevertheless, studying these processes using 15N-based stable isotope probing was so far limited because of technical challenges related to the relative rarity of nitrogen in nucleic acids and proteins compared to carbon, and because of its absence in lipids. However, the recent adoption of high-throughput sequencing and statistical modelling methods to SIP studies increased the sensitivity of the method and enabled overcoming some of the challenges. This chapter describes in detail how to perform DNA- and RNA-SIP using 15N.
- MeSH
- bakteriální RNA chemie genetika izolace a purifikace metabolismus MeSH
- bakterie fixující dusík genetika metabolismus MeSH
- centrifugace - gradient hustoty MeSH
- DNA bakterií chemie genetika izolace a purifikace metabolismus MeSH
- fixace dusíku genetika fyziologie MeSH
- izotopové značení metody MeSH
- izotopy dusíku metabolismus MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is considered to be a potential zoonotic pathogen and meat is one of the sources of MAP exposure for humans. MAP has been shown to be relatively resistant to different food processing methods, but there is a lack of information about the effects of ripening and fermentation processes on MAP survival in meat. Our results demonstrate that a short ripening process during teewurst production did not reduce MAP counts and viable mycobacteria were detected even during 4 weeks of storage. Although no viable MAP was recovered during the dry fermented sausage production process, there was no reduction in MAP count detected by real time PCR during production and storage of both sausages. Although the impact of foodborne exposure to viable MAP and/or mycobacterial components has not yet been clearly determined, the consumption of raw fermented meat products may be considered as a possible route of MAP transmission to humans.
- MeSH
- DNA bakterií izolace a purifikace MeSH
- fermentace MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- manipulace s potravinami metody MeSH
- masné výrobky mikrobiologie MeSH
- Mycobacterium avium subsp. paratuberculosis genetika růst a vývoj izolace a purifikace MeSH
- paratuberkulóza mikrobiologie MeSH
- prasata MeSH
- skladování potravin MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The efficiency of solid phase extraction (SPE) of DNA on polymer particles is limited by the features of the applied solid support, such as size, hydrophilicity, and functionality and their application in SPE also requires additional steps and compounds to finally obtain sufficient amount of high-quality DNA. The present study describes a preparation of sub-micrometer monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) (PME) particles by precipitation polymerization. The effect of the ethylene dimethacrylate (EDMA) crosslinker concentration on morphology and particle size, which varied from 730 to 900 nm, was investigated. The particles with 5 and 15 wt% EDMA were selected for a study of SPE of plasmid DNA under various adsorption and elution conditions, followed by the enzymatic restriction of isolated DNA to verify a quality the nucleic acid. The particles with 15 wt% EDMA were suitable for the SPE because they retained better colloidal stability during the adsorption without additional induction of DNA conformational change. The quality of isolated DNA was finally verified by enzymatic restriction by restriction endonuclease EcoRI. Moreover, the developed method using PME particles was successfully utilized for DNA isolation from Escherichia coli lysate.
- MeSH
- DNA bakterií chemie izolace a purifikace MeSH
- DNA chemie izolace a purifikace MeSH
- extrakce na pevné fázi * metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- koncentrace vodíkových iontů MeSH
- polymery chemie MeSH
- polymethylmethakrylát chemie MeSH
- velikost částic MeSH
- Publikační typ
- časopisecké články MeSH
Environmental matrices and food products are hypothesized to be sources of Cronobacter spp. The severity of neonatal infections, increasing number of cases in elderly and immunocompromised individuals, as well as isolation of Cronobacter spp. from clinical materials demands that more attention should be paid to Cronobacter spp. detection and occurrence of the bacteria in food products. Here, a total of 175 samples of ready-to-eat vegetables, frozen vegetables, and sprouted seeds were collected during a period of 1 year and examined for the presence of Cronobacter spp. using a cultivation method with two different sample preparations and real-time polymerase chain reaction (qPCR). In total, Cronobacter spp. were detected in 22.3% of tested samples using cultivation. In comparison, direct qPCR detected Cronobacter spp. in 37.7% of these samples (p < 0.01; Fisher's exact test) and the numbers of genome equivalents per gram reached 108 in some samples of sprouts. Cronobacter spp. were isolated from 51.4%, 37.2%, and 5.2% samples of sprouts, frozen vegetables, and cut green leaves/salads, respectively. Using qPCR, the most frequently contaminated sample types were sprouts (91.4%) and frozen vegetables (60.5%), whereas the rate of positivity for cut green leaves/salads was, in comparison, only 8.2% (p < 0.01; χ2 -test for independence). PRACTICAL APPLICATION: This study provided valuable information on the occurrence of Cronobacter spp. in ready-to-eat vegetables using cultivation and qPCR. Cronobacter spp. are emerging opportunistic pathogens that can be present in food of plant origin. Cronobacter spp. were isolated from sprouts, frozen vegetables, and cut green leaves/salads, and the numbers of genome equivalents per gram reached 108 in some samples of sprouts.
- MeSH
- Cronobacter izolace a purifikace MeSH
- DNA bakterií izolace a purifikace MeSH
- kontaminace potravin analýza MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- potravinářská mikrobiologie MeSH
- průmyslově zpracované potraviny mikrobiologie MeSH
- rostlinné produkty mikrobiologie MeSH
- zelenina mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
