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Background: The spread of coronavirus disease 2019 (COVID-19) in health care settings endangers patients with cancer. As knowledge of the transmission of COVID-19 emerged, strategies for preventing nosocomial COVID-19 were updated. We describe our early experience with nosocomial respiratory viral infections (RVIs) at a cancer center in the first year of the pandemic (March 2020-March 2021)., Methods: Nosocomial RVIs were identified through our infection control prospective surveillance program, which conducted epidemiologic investigations of all microbiologically documented RVIs. Data was presented as frequencies and percentages or medians and ranges., Results: A total of 35 of 3944 (0.9%) documented RVIs were determined to have been nosocomial acquired. Majority of RVIs were due to SARS CoV-2 (13/35; 37%) or by rhinovirus/enterovirus (12/35; 34%). A cluster investigation of the first 3 patients with nosocomial COVID-19 determined that transmission most likely occurred from employees to patients. Five patients (38%) required mechanical ventilation and 4 (31%) died during the same hospital encounter., Conclusions: Our investigation of the cluster led to enhancement of our infection control measures. The implications of COVID-19 vaccination on infection control policies is still unclear and further studies are needed to delineate its impact on the transmission of COVID-19 in a hospital setting., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis . As PagR plays a crucial role in the regulation of the protective antigen gene pagA , which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Bacillus subtilis, in its natural habitat, is regularly exposed to rapid changes in the osmolarity of its surrounding. As its primary survival strategy, it accumulates large amounts of the compatible solute proline by activating the de novo proline biosynthesis pathway and exploiting the glutamate pools. This osmotically-induced biosynthesis requires activation of a SigA-type promoter that drives the expression of the proHJ operon. Population-wide studies have shown that the activity of the proHJ promoter correlates with the increased osmotic pressure of the environment. Therefore, the activation of the proHJ transcription should be an adequate measure of the adaptation to osmotic stress through proline synthesis in the absence of other osmoprotectants. In this study, we investigate the kinetics of the proHJ promoter activation and the early adaptation to mild osmotic upshift at the single-cell level. Under these conditions, we observed a switching point and heterogeneous proline biosynthesis gene expression, where the subpopulation of cells showing active proHJ transcription is able to continuously divide, and those unresponsive to osmotic stress remain dormant. Additionally, we demonstrate that bactericidal antibiotics significantly upregulate proHJ transcription in the absence of externally imposed osmotic pressure, suggesting that the osmotically-controlled proline biosynthesis pathway is also involved in the antibiotic-mediated stress response., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure at low doses remain largely unexplored. Natural substances, such as plant-based secondary metabolites, are widely known for their effective antibacterial potential, which makes them promising as ingredients in food and also as a surrogate for antibiotics. In this work, we tested a range of structurally related phytochemicals, including benzene derivatives, monoterpenes, hydroxycinnamic acid derivatives and vitamins, for their inhibitory effects on the growth of B. cereus and the production of cereulide toxin. For this purpose, we developed a high-throughput, small-scale method which allowed us to analyze B. cereus survival and cereulide production simultaneously in one workflow by coupling an AlamarBlue-based viability assay with ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). This combinatory method allowed us to identify not only phytochemicals with high antibacterial potential, but also ones specifically eradicating cereulide biosynthesis already at very low concentrations, such as gingerol and curcumin.

Background: Cases of novel coronavirus disease 2019 (COVID-19) were first reported in Wuhan, China, in December 2019. In this report, we describe 3 clusters of COVID-19 infections among healthcare workers (HCWs), not associated with patient exposure, and the interventions undertaken to halt ongoing exposure and transmission at our cancer center., Methods: A cluster of cases was defined as 2 or more cases of severe acute respiratory coronavirus virus 2 (SARS-CoV-2)-positive COVID-19 among HCWs who work in the same unit area at the same time. Cases were identified by real-time reverse transcription polymerase chain reaction testing. Contact tracing, facility observations, and infection prevention assessments were performed to investigate the 3 clusters between March 1 and April 30, 2020, with subsequent implementation of containment strategies., Results: The initial cluster involved HCWs from an ancillary services unit, with contacts traced back to a gathering in a break room in which 1 employee was symptomatic, although not yet diagnosed with COVID-19, with subsequent transmission to 7 employees. The second cluster involved 4 employees and was community related. The third cluster involved only 2 employees with possible transmission while working in the same office at the same time. A step-up approach was implemented to control the spread of infection among employees, including universal masking, enhanced cleaning, increase awareness, and surveillance testing. No nosocomial transmission to patients transpired., Conclusions: To our knowledge, this is the first report of a hospital-based cluster of COVID-19 infections among HCWs in a cancer hospital describing our steps to mitigate further transmission.

Biofilm formation is a strategy of many bacterial species to adapt to a variety of stresses and has become a part of infections, contaminations, or beneficial interactions. In this study, we demonstrate that profound physiological changes permit Bacillus cereus to switch from a floating to a sessile lifestyle, to undergo further maturation of the biofilm and to differentiate into the offensive or defensive features. We report that floating and biofilm cells are populations that differentiate metabolically, with members of each subpopulation developing different branches of certain metabolic pathways. Secondly, biofilm populations rearrange nucleotides, sugars, amino acids, and energy metabolism. Thirdly, this metabolic rearrangement coexists with: the synthesis of the extracellular matrix, sporulation, reinforcement of the cell wall, activation of the ROS detoxification machinery and production of secondary metabolites. This strategy contributes to defend biofilm cells from competitors. However, floating cells maintain a fermentative metabolic status that ensures a higher aggressiveness against hosts, evidenced by the production of toxins. The maintenance of the two distinct subpopulations is an effective strategy to face different environmental conditions found in the life styles of B. cereus ., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been completely solved to achieve significant production-strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CodY and CcpA that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic mobility shift assay (EMSA) showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Tracking of fluorescent protein (FP)-labelled rhizobacteria is a key prerequisite to gain insights into plant-bacteria interaction mechanisms. However, the performance of FPs mostly has to be optimized for the bacterial host and for the environment of intended application. We report on the construction of mutational libraries of the superfolder green fluorescent protein sfGFP and the red fluorescent protein mKate2 in the bacterium B. mycoides, which next to its potential as plant-biocontrol agent occasionally enters an endophytic lifestyle. By fluorescence-activated cell sorting and comparison of signal intensities at the colony and single-cell level, the variants sfGFP(SPS6) and mKate (KPS12) with significantly increased brightness were isolated. Their high applicability for plant-bacteria interaction studies was shown by confocal laser scanning microscopy tracking of FP-tagged B. mycoides strains after inoculation to Chinese cabbage plants in a hydroponic system. During the process of colonization, strain EC18 rapidly attached to plant roots and formed a multicellular matrix, especially at the branching regions of the root hair, which probably constitute entrance sites to establish an endophytic lifestyle. The universal applicability of the novels FPs was proven by expression from a weak promoter, dual-labelling of B. mycoides, and by excellent expression and detectability in additional soil- and rhizosphere-associated Bacillus species., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius , an in vivo screening approach based on a mutational library of superfolder GFP was applied., Results: Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability., Conclusions: This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.

Plant root secreted compounds alter the gene expression of associated microorganisms by acting as signal molecules that either stimulate or repel the interaction with beneficial or harmful species, respectively. However, it is still unclear whether two distinct groups of beneficial bacteria, non-plant-associated (soil) strains and plant-associated (endophytic) strains, respond uniformly or variably to the exposure with root exudates. Therefore, Bacillus mycoides , a potential biocontrol agent and plant growth-promoting bacterium, was isolated from the endosphere of potatoes and from soil of the same geographical region. Confocal fluorescence microscopy of plants inoculated with GFP-tagged B. mycoides strains showed that the endosphere isolate EC18 had a stronger plant colonization ability and competed more successfully for the colonization sites than the soil isolate SB8. To dissect these phenotypic differences, the genomes of the two strains were sequenced and the transcriptome response to potato root exudates was compared. The global transcriptome profiles evidenced that the endophytic isolate responded more pronounced than the soil-derived isolate and a higher number of significant differentially expressed genes were detected. Both isolates responded with the alteration of expression of an overlapping set of genes, which had previously been reported to be involved in plant-microbe interactions; including organic substance metabolism, oxidative reduction, and transmembrane transport. Notably, several genes were specifically upregulated in the endosphere isolate EC18, while being oppositely downregulated in the soil isolate SB8. These genes mainly encoded membrane proteins, transcriptional regulators or were involved in amino acid metabolism and biosynthesis. By contrast, several genes upregulated in the soil isolate SB8 and downregulated in the endosphere isolate EC18 were related to sugar transport, which might coincide with the different nutrient availability in the two environments. Altogether, the presented transcriptome profiles provide highly improved insights into the life strategies of plant-associated endophytes and soil isolates of B. mycoides .

Identifying Bacillus cereus as the causative agent of a foodborne outbreak still poses a challenge. We report on the epidemiological and microbiological investigation of three outbreaks of food poisoning (A, B, and C) in Austria in 2013. A total of 44% among 32 hotel guests (A), 22% among 63 employees (B) and 29% among 362 residents of a rehab clinic (C) fell sick immediately after meal consumption. B. cereus isolated from left overs or retained samples from related foods were characterized by toxin gene profiling, and molecular typing using panC sequencing and M13-PCR typing (in outbreak A and C). We identified two B. cereus strains in outbreak A, and six B. cereus strains, each in outbreak B and C; we also found Staphylococcus aureus and staphylococcal enterotoxins in outbreak A. The panC sequence based phylogenetic affiliation of the B. cereus strains, together with findings of the retrospective cohort analyses, helped determining their etiological role. Consumption of a mashed potatoes dish in outbreak A (RR: ∞), a pancake strips soup in outbreak B (RR 13.0; 95% CI 1.8-93.0) and for outbreak C of a fruit salad (RR 1.50; 95% CI 1.09-2.00), deer ragout (RR: 1.99; 95% CI 1.23-3.22) and a cranberry/pear (RR 2.46; 95% CI 1.50-4.03)were associated with increased risk of falling sick. An enterotoxigenic strain affiliated to the phylogenetic group with the highest risk of food poisoning was isolated from the crème spinach and the strawberry buttermilk, and also from the stool samples of the one B. cereus positive outbreak case-patient, who ate both. Our investigation of three food poisoning outbreaks illustrates the added value of a combined approach by using epidemiological, microbiological and genotyping methods in identifying the likely outbreak sources and the etiological B. cereus strains., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Vaccination of health care workers (HCWs) remains a key strategy to reduce the burden of influenza infections in cancer patients., Methods: In this 8-year study, we evaluated the effect of a multifaceted approach, including a mandatory influenza vaccination program, on HCW vaccination rates and its effect on nosocomial influenza infections in cancer patients., Results: The influenza vaccination rate of all employees significantly increased from 56% (8,762/15,693) in 2006-2007 to 94% (17,927/19,114) in 2013-2014 (P < .0001). The 2009 mandatory participation program increased HCW vaccination rates in the targeted groups (P < .0001), and the addition of an institutional policy in 2012 requiring influenza vaccination or surgical mask use with each patient contact further increased vaccination rates by 10%-18% for all groups in 1 year. The proportion of nosocomial influenza infections significantly decreased (P = .045) during the study period and was significantly associated with increased HCW vaccination rates in the nursing staff (P = .043) and in personnel working in high-risk areas (P = .0497)., Conclusions: Multifaceted influenza vaccination programs supported by institutional policy effectively increased HCW vaccination rates. Increased HCW vaccination rates were associated with a reduction in the proportion of nosocomial influenza infections in immunocompromised cancer patients., (Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5' intergenic regions (5' IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5' IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5' untranslated regions (5' UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5' UTR in B. cereus INRA C3 showed that the entire 331 bp 5' UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5' UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5' IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5' UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are highly conserved among all B. cereus sensu lato strains, indicating that this regulator does not significantly contribute to the heterogeneity in virulence potentials. The CodY recognition sites are far less conserved, perhaps conferring varying strengths of CodY binding, which might modulate toxin synthesis in a strain-specific manner.

Unlabelled: Bacillus cereus is among the microorganisms most often isolated from cases of food spoilage and causes gastrointestinal diseases as well as nongastrointestinal infections elicited by the emetic toxin cereulide, enterotoxins, and a panel of tissue-destructive virulence factors. This opportunistic pathogen is increasingly associated with rapidly fatal clinical infections especially linked to neonates and immunocompromised individuals. Fatality results from either the misdiagnosis of B. cereus as a contaminant of the clinical specimen or from failure of antibiotic therapy. Here we report for the first time that exposure to aminoglycoside antibiotics induces a phenotype switching of emetic B. cereus subpopulations to a slow-growing small colony variant (SCV) state. Along with altered antibiotic resistance, SCVs showed distinct phenotypic and metabolic properties, bearing the risk of antibiotic treatment failure and of clinical misdiagnosis by standard identification tests used in routine diagnostic. The SCV subpopulation is characterized by enhanced production of the toxin cereulide, but it does not secrete tissue-destructive and immune system-affecting enzymes such as sphingomyelinase and phospholipase. SCVs showed significantly prolonged persistence and decreased virulence in the Galleria mellonella model for bacterial infections, indicating diversification concerning their ecological lifestyle. Importantly, diversification into coexisting wild-type and SCV subpopulations also emerged during amikacin pressure during in vivo infection experiments., Importance: This study shows for the first time that pathogenic spore-forming B. cereus strains are able to switch to a so far unreported slow-growing lifestyle, which differs substantially in terms of developmental, phenotypic, metabolic, and virulence traits from the wild-type populations. This underpins the necessity of molecular-based differential diagnostics and a well-chosen therapeutic treatment strategy in clinical environments to combat B. cereus in a tailored manner. The reported induction of SCV in an endospore-forming human pathogen requires further research to broaden our understanding of a yet unexplored antibiotic resistance mechanism in sporulating bacteria. Our work also raises a general question about the ecological meaning of SCV subpopulation emergence and importance of SCV in sporeformer populations as an alternative route, next to sporulation, to cope with stresses encountered in natural niches, such as soil or host interfaces., (Copyright © 2015 Frenzel et al.)

α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) β/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

Consumption of food products contaminated with cereulide (1), a toxin produced by Bacillus cereus, might cause intoxications with symptoms reported to range from indigestion pain and emesis to death. Recently, a series of structural variants, coined isocereulides A-G (2-8), were identified for the first time to be produced along with cereulide (1). The observation that isocereulide A (2) shows an ∼ 8-fold increased cytotoxicity when compared to 1 urges the development of analytical tools enabling an accurate quantitation of these toxins. Therefore, a rapid, sensitive, and robust stable isotope dilution assay (SIDA) was developed for the combined quantitation of 1-8 by means of UPLC-MS/MS. On average, trueness and precision of the method were 112.5 ± 1.8% RSD, repeatability and reproducibility were 2 and 4% for cereulide and isocereulides A-G, and the LOD and LOQ of 0.1 and 0.5 ng/g, respectively, demonstrated a high sensitivity for the developed SIDA method. Application of this method to food samples revealed elevated levels of 1-8 in two suspicious noodle samples, for example, ranging from 0.59 (7) to 189.08 ng/g (1) in sample 1 and from 5.77 (7) to 6198.17 ng/g (1) in sample 2, whereas the analysis of 25 randomly selected food samples, which have not been the subject to any complaints, did not contain detectable amounts of any of these toxins. As a consequence, this SIDA method could add an important contribution to the knowledge-based risk assessment of B. cereus toxins in foods.

Bacillus cereus is a Gram-positive endospore forming bacterium known for its wide spectrum of phenotypic traits, enabling it to occupy diverse ecological niches. Although the population structure of B. cereus is highly dynamic and rather panmictic, production of the emetic B. cereus toxin cereulide is restricted to strains with specific genotypic traits, associated with distinct environmental habitats. Cereulide is an ionophoric dodecadepsipeptide that is produced non-ribosomally by an enzyme complex with an unusual modular structure, named cereulide synthetase (Ces non-ribosomal peptide synthetase). The ces gene locus is encoded on a mega virulence plasmid related to the B. anthracis toxin plasmid pXO1. Cereulide, a highly thermo- and pH- resistant molecule, is preformed in food, evokes vomiting a few hours after ingestion, and was shown to be the direct cause of gastroenteritis symptoms; occasionally it is implicated in severe clinical manifestations including acute liver failures. Control of toxin gene expression in emetic B. cereus involves central transcriptional regulators, such as CodY and AbrB, thereby inextricably linking toxin gene expression to life cycle phases and specific conditions, such as the nutrient supply encountered in food matrices. While in recent years considerable progress has been made in the molecular and biochemical characterization of cereulide toxin synthesis, far less is known about the embedment of toxin synthesis in the life cycle of B. cereus. Information about signals acting on toxin production in the food environment is lacking. We summarize the data available on the complex regulatory network controlling cereulide toxin synthesis, discuss the role of intrinsic and extrinsic factors acting on toxin biosynthesis in emetic B. cereus and stress how unraveling these processes can lead to the development of novel effective strategies to prevent toxin synthesis in the food production and processing chain.

Cereulide and isocereulides A-G are biosynthesized as emetic toxins by Bacillus cereus via a non-ribosomal peptide synthetase (NRPS) called Ces. Although a thiotemplate mechanisms involving cyclo-trimerization of ready-made D-O-Leu-D-Ala-L-O-Val-L-Val via a thioesterase (TE) domain is proposed for cereulide biosynthesis, the exact mechanism is far from being understood. UPLC-TOF MS analysis of B. cereus strains in combination with (13)C-labeling experiments now revealed tetra-, octa-, and dodecapeptides of a different sequence, namely (L-O-Val-L-Val-D-O-Leu-D-Ala)1-3, as intermediates of cereulide biosynthesis. Surprisingly, also di-, hexa-, and decadepsipeptides were identified which, together with the structures of the previously reported isocereulides E, F, and G, do not correlate to the currently proposed mechanism for cereulide biosynthesis and violate the canonical NRPS biosynthetic logic. UPLC-TOF MS metabolite analysis and bioinformatic gene cluster analysis highlighted dipeptides rather than single amino or hydroxy acids as the basic modules in tetradepsipeptide assembly and proposed the CesA C-terminal C* domain and the CesB C-terminal TE domain to function as a cooperative esterification and depsipeptide elongation center repeatedly recruiting the action of the C* domain to oligomerize tetradepsipeptides prior to the release of cereulide from the TE domain by macrocyclization.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

Interleukin-3 (IL-3) is capable of supporting the proliferation of a broad range of hematopoietic cell types, whereas granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) represent critical cytokines in myeloid differentiation. When this was investigated in a pluripotent-stem-cell-based hematopoietic differentiation model, IL-3/G-CSF or IL-3/M-CSF exposure resulted in the continuous generation of myeloid cells from an intermediate myeloid-cell-forming complex containing CD34(+) clonogenic progenitor cells for more than 2 months. Whereas IL-3/G-CSF directed differentiation toward CD45(+)CD11b(+)CD15(+)CD16(+)CD66b(+) granulocytic cells of various differentiation stages up to a segmented morphology displaying the capacity of cytokine-directed migration, respiratory burst response, and neutrophil-extracellular-trap formation, exposure to IL-3/M-CSF resulted in CD45(+)CD11b(+)CD14(+)CD163(+)CD68(+) monocyte/macrophage-type cells capable of phagocytosis and cytokine secretion. Hence, we show here that myeloid specification of human pluripotent stem cells by IL-3/G-CSF or IL-3/M-CSF allows for prolonged and large-scale production of myeloid cells, and thus is suited for cell-fate and disease-modeling studies as well as gene- and cell-therapy applications., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14(+) monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14(+) monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation., (Copyright © 2014 Elsevier Inc. All rights reserved.)