Automated detection and enumeration of fluorescently labelled bacteria with flow cytometry

Rapid identification of specific microorganisms in their natural environments is of high importance when their cultivation is either impossible or cannot provide valid results. One of the tools available for such purposes is flow-FISH where classical microscopic identification of microorganisms is combined with an automated enumeration system. Despite the high potential of flow-FISH there are still many unsolved issues to introduce the method. The aim of this research was to determine potential quantification limits of a simple flow-FISH protocol to ensure specific and rapid automated identification of target cells.The results of the study showed that at optimal hybridization conditions (16 hours of hybridization, 15 minutes post-hybridization washing and 3 ng/µl probe concentration) it is possible to specifically determine all main proteobacteria groups and Grampositive bacteria and discriminate among β and γ proteobacteria. Detection of α-proteobacteria was not achieved in this study. Despite the promising application potential of flow-FISH, high attention must be made to extensive cell loss (up to 59%) during preparation of samples for the analyses

Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications

BACKGROUND: Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage. RESULTS: Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the λimm434 prophage. CONCLUSIONS: Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains

Anti-biofilm Activity of Antibody Directed Against Surface Antigen Complement Receptor 3-Related Protein-Comparison of Candida Albicans and Candida Dubliniensis

Candida species (spp.) are a part of the normal human microbiota. Candida dubliniensis mostly colonizes the oral cavity and/or respiratory tract (Mahelová and Růžička 2017), especially in HIV-infected individuals (Coleman et al.1997; Sullivan et al.2004; Wahab et al.2014), while Candida albicans is a common inhabitant of the gastrointestinal tract, urogenital tract and oral cavity (Sardi et al.2013; Höfs, Mogavero and Hube 2016). Candidiasis is the most common global fungal infection (Sardi et al.2013). Candida albicans has been isolated in more than 50% of candidiasis; however, the number of non-albicans spp. able to cause serious candidiasis has increased in recent years (Yapar 2014; Pu et al.2015; Sandhu et al.2017). Although C. dubliniensis is phylogenetically very similar to C. albicans, it differs in some genes, especially those coding for virulence-associated proteins. Candida dubliniensis lacks more than 168 genes characteristic of its ‘yeast-cousin’ C. albicans (Jackson et al.2009), the majority of them encoding proteins related to the yeast-to-hyphae transition, tissue invasion or biofilm development (Moran et al.2004; Jackson et al.2009; Moran, Coleman and Sullivan 2012). Moreover, C. dubliniensis manifests a higher predisposition to develop resistance to fluconazole (Sullivan et al.1995; Moran, Coleman and Sullivan 2012; Jordan et al.2014). On the other hand, both C. albincans and C. dubliniensis are able to form a biofilm (Sullivan et al.2004; Borghi et al.2014). Adherence is the first and most crucial step in biofilm development, and various surface antigens participate in this process (Chaffin 2008; Gow and Hube 2012; Hebecker et al.2014). CR3-RP (complement receptor 3-related protein) is one of the cell surface antigens of Candida spp. with functional and structural similarity to the human complement receptor 3 (CR3) expressed on neutrophils, macrophages and monocytes. CR3-RP has been demonstrated to bind human complement fragment iC3b and to mediate leukocyte diapedesis (Heidenreich and Dierich 1985; Bujdáková et al.1997). Additionally, CR3-RP seems to be an important immunogenic mannoprotein participating in adhesion and biofilm development (Bujdáková et al.2008, 2010). A fragment of CR3-RP was sequenced (DINGGGATLPQ), and according to this sequence, CR3-RP was categorized into the DING protein family (named after DINGGG N termini) (Bujdáková et al.2008; Bernier 2013). Some other surface proteins contributing to biofilm development have been described, such as Eap protein, the Als protein family, the Hwp1 or MP65 proteins (Gomez et al.1996; Nailis et al.2010; Finkel and Mitchell 2011; Araújo, Henriques and Silva 2017). Additionally, antibodies generated after the immunization of animals with some of the above proteins seems to be promising in tools focused on fighting yeast infections (Fujibayashi et al.2009; Mishra, Ali and Shukla 2015; Torosantucci et al.2017). Recent studies showed that antibodies targeting Als3 (Coleman et al.2009), MP65 (De Bernardis et al.2007) or another 42.7 kDa unnamed surface antigen in the Candida cell wall (Mishra, Ali and Shukla 2015) decreased adhesion and biofilm formation

Investigation of mechanisms involved in germination enhancement of wheat (Triticum aestivum) by cold plasma: Effects on seed surface chemistry and characteristics

Recent reports indicate that atmospheric cold plasma (ACP) treatment of seeds can enhance their germination, however, the mechanisms of action are not yet entirely clear. In the present work, we report on the effects of plasma treatment on wheat seed germination and seedling growth. Additionally, changes in the surface chemistry and characteristics of the wheat seeds exposed to plasma were investigated. Treatments of 30–60 s significantly enhanced the germination rate and showed positive effects on seedling growth. ACP resulted in changes of seed surface and chemical characteristics including water uptake and contact angle values. Changes in seed pH and total titratable acidity, as well as nitrites, nitrates, and malondialdehyde concentrations were also recorded

The potential of atmospheric air cold plasma for control of bacterial contaminants relevant to cereal grain production

The aim of this work was to investigate the efficacy of dielectric barrier discharge atmospheric cold plasma (DBD ACP) against bacteria associated with grains quality and safety. ACP inactivation efficacy was tested against biofilms formed by different strains of E. coli, Bacillus and Lactobacillus in grain model media and against B. atrophaeus endospores either in grain media or attached on abiotic surfaces. Effects were dependent on bacterial strain, media composition and mode of ACP exposure. ACP treatment for 5min reduced E. coli spp., B. subtilis and Lactobacillus spp. biofilms by \u3e3 log10, whereas insignificant reductions were achieved for B. atrophaeus. ACP treatment of 5–20min reduced B. atrophaeus spores in liquids by \u3e5 log10. Treatment for 30min reduced spores on hydrophobic surface by \u3e6 log10, whereas maximum of 4.4 log reductions were achieved with spores attached to hydrophilic surface. Microscopy demonstrated that ACP caused significant damage to spores. In package ACP treatment has potential to inactivate grain contaminants in the form of biofilms, as well as spores and vegetative cells. Industrial relevance This study demonstrates that ACP technology is a promising tool for effective bio-decontamination which offers a wide range of possible applications including inactivation of microorganisms on cereal grains. However, due to the nature of the microbial contamination of grains and complex grain structures it may be necessary to optimise the potential for surface inactivation at several stages of grain processing and storage to enhance ACP efficacy against bacterial endospores

Influence of Exogenously Supplemented Caffeine on Cell Division, Germination, and Growth of Economically Important Plants

Caffeine is a plant secondary metabolite of antiherbivory, allelopathic, and antibacterial activity. In our previous study, caffeine was shown to be an effective agent toward plant pathogenic bacteria causing high economic losses in crop production worldwide. Current study indicated that growth media supplementation with soil or plant extract did not interfere with antibacterial action of caffeine against Clavibacter michiganensis, Dickeya solani, Pectobacterium atrosepticum, Pectobacterium carotovorum, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas campestris. The impact of caffeine on plant cell division, seed germination and growth of economically important plants was evaluated to assess possible applicability of caffeine in plant protection field. Caffeine impaired plant cell division process and inhibited in vitro germination of tomato and lettuce. Regeneration of potato explants was also negatively affected by the addition of caffeine. However, caffeine spraying or watering of tomato, lettuce and cabbage grown in soil did not impair plant development. Although the tested plants accumulated caffeine, its inner quantity was reduced by peeling and/or cooking. According to the results, caffeine warrants additional attention as a useful, natural compound designated for the control of bacterial plant pathogens. Proposed treatment seems promising especially in the case of providing protection for overwinter-stored table potato tubers

Plasma activated water and airborne ultrasound treatments for enhanced germination and growth of soybean

The effect of two novel technologies, also in combination, on germination and growth of soybeans has been investigated. On one side, ultrasound treatment of the seeds increased water uptake without altering the morphology and the wettability of the seed coat, but also induced slight chemical modifications of the outer part of the seed. Plasma-activated water (PAW), obtained from treating water with non-thermal atmospheric-pressure plasma in air, increased the rate of germination and subsequent plant growth. Different combinations of these two technologies were tested in order to study their interaction and to identify an optimum treatment process. Industrial relevance A great urgency in crop management is to enhance sustainability. The aim is to achieve a cheap and eco-friendly production process reducing the wide current use of energy, irrigation water, chemicals and pesticides. Soybeans is a legume whose worldwide production is increasing in the last years therefore a higher efficiency and sustainability in its cultivation is obviously very appealing. Cold plasma and Ultrasound technologies are well-known in the industrial scenario and their applications in crop production are recently drawing attention; the potential of combining these two powerful techniques is clearly very promising

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362