Circadian regulation of metabolic, cell division, and cation transport promoters in the gastrointestinal bacterium Klebsiella aerogenes.

Introduction: All eukaryotes and at least some prokaryotes express the capacity to anticipate and adapt to daily changes of light and temperature in their environments. These circadian programs are fundamental features of many forms of life. Cyanobacteria were the first prokaryotes to have demonstrated circadian gene expression. Recently, a circadian rhythm was also discovered in an unrelated bacterium, Klebsiella aerogenes, a human gut commensal and nosocomial pathogen. Methods: Here we characterize new clock-controlled genes with spatial differences in expression using a bacterial luciferase reporter. These include dephospho-coenzyme A kinase (coaE), manganese transporter, H-dependent (mntH) and a gene identified as filamenting temperature-sensitive mutant Z (ftsZ). Results and Discussion: The data show that all three reporter constructs exhibited circadian variation, although only PmntH::luxCDABE reporter strains were synchronized by melatonin. Additionally, we show that K. aerogenes divides rhythmically in vitro and that these bacteria may alternate between exponential and stationary cells. Together, these findings provide a deeper understanding of K. aerogenes.

Transcriptional effects of melatonin on the gut commensal bacterium Klebsiella aerogenes.

Klebsiella (nee Enterobacter) aerogenes is the first human gut commensal bacterium with a documented sensitivity to the pineal/gastrointestinal hormone melatonin. Exogenous melatonin specifically increases the size of macrocolonies on semisolid agar and synchronizes the circadian clock of K. aerogenes in a concentration dependent manner. However, the mechanisms driving these phenomena are unknown. In this study, we applied RNA sequencing to identify melatonin sensitive transcripts during culture maturation. This work demonstrates that the majority of melatonin sensitive genes are growth stage specific. Melatonin exposure induced differential gene expression of 81 transcripts during exponential growth and 30 during early stationary phase. This indole molecule affects genes related to biofilm formation, fimbria biogenesis, transcriptional regulators, carbohydrate transport and metabolism, phosphotransferase systems (PTS), stress response, metal ion binding and transport. Differential expression of biofilm and fimbria-related genes may be responsible for the observed differences in macrocolony area. These data suggest that melatonin enhances Klebsiella aerogenes host colonization.

Entrainment of the Circadian Clock of the Enteric Bacterium Klebsiella aerogenes by Temperature Cycles.

The gastrointestinal bacterium Klebsiella (née Enterobacter) aerogenes expresses an endogenously generated, temperature-compensated circadian rhythm in swarming motility. We hypothesized that this rhythm may be synchronized/entrained in vivo by body temperature (TB). To determine entrainment, cultures expressing bioluminescence were exposed to temperature cycles of 1°C (35°C-36°C) or 3°C (34°C-37°C) in amplitude at periods (T-cycles) of T = 22, T = 24, or T = 28 h. Bacteria entrained to all T-cycles at both amplitudes and with stable phase relationships. A high-amplitude phase response curve (PRC) in response to 1-h pulses of 3°C temperature spike (34°C-37°C) at different circadian phases was constructed, revealing a Type-0 phase resetting paradigm. Furthermore, real-time bioluminescence imaging revealed a spatiotemporal pattern to the circadian rhythm. These data are consistent with the hypothesis that the K. aerogenes circadian clock entrains to its host via detection of and phase shifting to the daily pattern of TB.

Sensitive Flow-through Immunoassay for Rapid Multiplex Determination of Cereal-borne Mycotoxins in Feed and Feed Ingredients.

An easy-to-operate membrane-based flow-through test for multiplex screening of four mycotoxins (zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A) in a variety of cereal-based feed ingredients and compound feeds, such as wheat, barley, soybean, wheat bran, rice, rice bran, maize, rapeseed meal, and sunflower meal, and various types of complete feed (duckling feed, swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were evaluated by enzyme-linked immunosorbent assay and then employed in the membrane rapid test. The cutoff levels for zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A were 50, 200, 1, and 10 µg/kg, respectively, based on European regulations and consumers' requirements. As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by addition of C18 sorbent, and final filtration of supernatant were followed. Both the sample preparation and the analysis procedure were simple, cost-effective, and easy to perform on-site in a nonlaboratory environment. The impact of sample processing on the result of the experiment was investigated supported by experimental design. The validation procedure was performed on the basis of Commission Regulation 2006/401/EC. The numbers of false-positive and false-negative outcomes were <5%, going along with the Commission Decision 2002/657/EC. Liquid chromatography-tandem mass spectrometry was performed as a confirmatory technique.

Capacitive sensing of N-formylamphetamine based on immobilized molecular imprinted polymers.

A highly sensitive, capacitive biosensor was developed to monitor trace amounts of an amphetamine precursor in aqueous samples. The sensing element is a gold electrode with molecular imprinted polymers (MIPs) immobilized on its surface. A continuous-flow system with timed injections was used to simulate flowing waterways, such as sewers, springs, rivers, etc., ensuring wide applicability of the developed product. MIPs, implemented as a recognition element due to their stability under harsh environmental conditions, were synthesized using thermo- and UV-initiated polymerization techniques. The obtained particles were compared against commercially available MIPs according to specificity and selectivity metrics; commercial MIPs were characterized by quite broad cross-reactivity to other structurally related amphetamine-type stimulants. After the best batch of MIPs was chosen, different strategies for immobilizing them on the gold electrode's surface were evaluated, and their stability was also verified. The complete, developed system was validated through analysis of spiked samples. The limit of detection (LOD) for N-formyl amphetamine was determined to be 10µM in this capacitive biosensor system. The obtained results indicate future possible applications of this MIPs-based capacitive biosensor for environmental and forensic analysis. To the best of our knowledge there are no existing MIPs-based sensors toward amphetamine-type stimulants (ATS).

Metabolism of modified mycotoxins studied through in vitro and in vivo models: an overview.

Mycotoxins are toxic, secondary metabolites produced by fungi. They occur in a wide variety of food and feed commodities, and are of major public health concern because they are the most hazardous of all food and feed contaminants in terms of chronic toxicity. In the past decades, it has become clear that in mycotoxin-contaminated commodities, many structurally related compounds generated by plant metabolism, fungi or food processing coexist with their free mycotoxins, defined as modified mycotoxins. These modified xenobiotics might endanger animal and human health as they are possibly hydrolysed into their free toxins in the digestive tract of mammals, and may consequently contribute to an unexpected high toxicity. As modified toxins represent an emerging issue, it is not a surprise that for most toxicological tests data are scarce to non-existent. Therefore, there is a need to elucidate the disposition and kinetics of both free and modified mycotoxins in mammals to correctly interpret occurrence data and biomonitoring results. This review emphasizes the current knowledge on the metabolism of modified mycotoxins using in vitro and in vivo models.