Gut microbiota-derived butyrate selectively interferes with growth of carbapenem-resistant Escherichia coli based on their resistance mechanism.

We investigated consequences of resistance acquisition in Escherichia coli clinical isolates during anaerobic (continuous culture) growth and examined their sensitivity to butyrate, a hallmark metabolite of healthy gut microbiota. Strains were stratified based on carrying either a carbapenemase (CARB) or displaying porin malfunctioning (POR). POR displayed markedly altered growth efficiencies, lower membrane stability and increased sensitivity to butyrate compared with CARB. Major differences in global gene expression between the two groups during anaerobic growth were revealed involving increased expression of alternative substrate influx routes, the stringent response and iron acquisition together with lower expression of various stress response systems in POR. Longitudinal analyses during butyrate wash-in showed common responses for all strains as well as specific features of POR that displayed strong initial "overshoot" reactions affecting various stress responses that balanced out over time. Results were partly reproduced in a mutant strain verifying porin deficiencies as the major underlying mechanism for results observed in clinical isolates. Furthermore, direct competition experiments confirmed butyrate as key for amplifying fitness disadvantages based on porin malfunctioning. Results provide new (molecular) insights into ecological consequences of resistance acquisition and can assist in developing measures to prevent colonization and infection based on the underlying resistance mechanism.

Enrichment of Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Strictly Anaerobic Sulfate-Reducing Cultures from Contaminated Soil and Sediment.

Sulfate-reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH-degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH-degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH-degrading SRB cultures. In this set of protocols, we describe step-by-step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate-reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow-growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography-mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium-throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH-degrading sulfate-reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Obtaining PAH-degrading strictly anaerobic sulfate-reducing enrichment cultures from contaminated soil and sediment Support Protocol 1: Operation and maintenance of an anaerobic workstation Support Protocol 2: Setup of gas purging systems for preparing anoxic solutions Support Protocol 3: Verification of viability in slow-growing SRB enrichment cultures Support Protocol 4: Extraction of genomic DNA from low-biomass cultures Basic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC-MS Basic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB cultures Basic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue method Alternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method.

Predicting butyrate- and propionate-forming bacteria of gut microbiota from sequencing data.

The bacteria-derived short-chain fatty acids (SCFAs) butyrate and propionate play important (distinct) roles in health and disease, and understanding the ecology of respective bacteria on a community-wide level is a top priority in microbiome research. Applying sequence data (metagenomics and 16S rRNA gene) to predict SCFAs production in vitro and in vivo, a clear split between butyrate- and propionate-forming bacteria was detected with only very few taxa exhibiting pathways for the production of both SCFAs. After in vitro growth of fecal communities from distinct donors (n = 8) on different substrates (n = 7), abundances of bacteria exhibiting pathways correlated with respective SCFA concentrations, in particular in the case of butyrate. For propionate, correlations were weaker, indicating that its production is less imprinted into the core metabolism compared with butyrate-forming bacteria. Longitudinal measurements in vivo (n = 5 time-points from 20 subjects) also revealed a correlation between abundances of pathway-carrying bacteria and concentrations of the two SCFAs. Additionally, lower bacterial cell concentrations, together with higher stool moisture, promoted overall bacterial activity (measured by flow cytometry and coverage patterns of metagenome-assembled genomes) that led to elevated SCFA concentrations with over-proportional levels of butyrate. Predictions on pathway abundances based on 16S rRNA gene data using our in-house database worked well, yielding similar results as metagenomic-based analyses. Our study indicates that stimulating growth of butyrate- and propionate-producing bacteria directly leads to more production of those compounds, which is governed by two functionally distinct bacterial groups facilitating the development of precision intervention strategies targeting either metabolite.

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities.

Microbiome research needs comprehensive repositories of cultured bacteria from the intestine of mammalian hosts. We expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes strain-level diversity, small-sized bacteria, and previously undescribed taxa (one family, 10 genera, and 39 species). This collection enabled metagenome-educated prediction of synthetic communities (SYNs) that capture key functional differences between microbiomes, notably identifying communities associated with either resistance or susceptibility to DSS-induced colitis. Additionally, nine species were used to amend the Oligo-Mouse Microbiota (OMM)12 model, yielding the OMM19.1 model. The added strains compensated for phenotype differences between OMM12 and specific pathogen-free mice, including body composition and immune cells in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available for future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

A protocol for the cultivation and monitoring of ileal gut microbiota surrogates.

AIMS: This research aimed to develop and validate a cultivation and monitoring protocol that is suitable for a surrogate microbial community that accounts for the gut microbiota of the ileum of the small intestine. METHODS AND RESULTS: Five bacterial species have been selected as representatives of the ileal gut microbiota and a general anaerobic medium (MS-BHI, as minimally supplemented brain heart infusion) has been constructed and validated against BCCM/LGM recommended and commercial media. Moreover, appropriate selective/differential media have been investigated for monitoring each ileal gut microbiota surrogate. Results showed that MS-BHI was highly efficient in displaying individual and collective behaviour of the ileal gut microbiota species, when compared with other types of media. Likewise, the selective/differential media managed to identify and describe the behaviour of their targeted species. CONCLUSIONS: MS-BHI renders a highly efficient, inexpensive and easy-to-prepare cultivation and enumeration alternative for the surrogate ileal microbiota species. Additionally, the selective/differential media can identify and quantify the bacteria of the surrogate ileal microbial community. SIGNIFICANCE AND IMPACT OF STUDY: The selected gut microbiota species can represent an in vitro ileal community, forming the basis for future studies on small intestinal microbiota. MS-BHI and the proposed monitoring protocol can be used as a standard for gut microbiota studies that utilize conventional microbiological techniques.

Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink.

Glycerol has become an attractive substrate for bio-based production processes. However, Escherichia coli, an established production organism in the biotech industry, is not able to grow on glycerol under strictly anaerobic conditions in defined minimal medium due to redox imbalance. Despite extensive research efforts aiming to overcome these limitations, anaerobic growth of wild-type E. coli on glycerol always required either the addition of electron acceptors for anaerobic respiration (e.g. fumarate) or the supplementation with complex and relatively expensive additives (tryptone or yeast extract). In the present work, driven by model-based calculations, we propose and validate a novel and simple strategy to enable fermentative growth of E. coli on glycerol in defined minimal medium. We show that redox balance could be achieved by uptake of small amounts of acetate with subsequent reduction to ethanol via acetyl-CoA. Using a directed laboratory evolution approach, we were able to confirm this hypothesis and to generate an E. coli strain that shows, under anaerobic conditions with glycerol as the main substrate and acetate as co-substrate, robust growth (µ = 0.06 h-1), a high specific glycerol uptake rate (10.2 mmol/gDW/h) and an ethanol yield close to the theoretical maximum (0.92 mol per mol glycerol). Using further stoichiometric calculations, we also clarify why complex additives such as tryptone used in previous studies enable E. coli to achieve redox balance. Our results provide new biological insights regarding the fermentative metabolism of E. coli and offer new perspectives for sustainable production processes based on glycerol.

Study of Fe-S Cluster Proteins in Methanococcus maripaludis , a Model Archaeal Organism.

Iron-sulfur (Fe-S) clusters are among the oldest and most versatile cofactors present in all domains of life. Many bacterial and eukaryotic Fe-S proteins have been well-characterized, whereas the archaeal ones are less studied. Fe-S proteins are particularly abundant and play essential roles in methanogenic archaea. Methanococcus maripaludis is a model methanogen with available genetic tools. Here, we describe the techniques for anaerobic cultivation of M. maripaludis with formate, liposome-mediated transformation, expression and anoxic affinity purification of Fe-S proteins, Fe-S cluster reconstitution, and analysis of Fe-S proteins by UV-visible absorption spectroscopy.

Towards the Anaerobic Production of Surfactin Using Bacillus subtilis.

The anaerobic growth of B. subtilis to synthesize surfactin poses an alternative strategy to conventional aerobic cultivations. In general, the strong foam formation observed during aerobic processes represents a major obstacle. Anaerobic processes have, amongst others, the distinct advantage that the total bioreactor volume can be exploited as foaming does not occur. Recent studies also reported on promising product per biomass yields. However, anaerobic growth in comparison to aerobic processes has several disadvantages. For example, the overall titers are comparably low and cultivations are time-consuming due to low growth rates. B. subtilis JABs24, a derivate of strain 168 with the ability to synthesize surfactin, was used as model strain in this study. Ammonium and nitrite were hypothesized to negatively influence anaerobic growth. Ammonium with initial concentrations up to 0.2 mol/L was shown to have no significant impact on growth, but increasing concentrations resulted in decreased surfactin titers and reduced nitrate reductase expression. Anaerobic cultivations spiked with increasing nitrite concentrations resulted in prolonged lag-phases. Indeed, growth rates were in a similar range after the lag-phase indicating that nitrite has a neglectable effect on the observed decreasing growth rates. In bioreactor cultivations, the specific growth rate decreased with increasing glucose concentrations during the time course of both batch and fed-batch processes to less than 0.05 1/h. In addition, surfactin titers, overall Y P/X and Y P/S were 53%, ∼42%, and ∼57% lower than in serum flask with 0.190 g/L, 0.344 g/g and 0.015 g/g. The Y X/S, on the contrary, was 30% lower in the serum flask with 0.044 g/g. The productivities q were similar with ∼0.005 g/(g⋅h). However, acetate strongly accumulated during cultivation and was posed as further metabolite that might negatively influence anaerobic growth. Acetate added to anaerobic cultivations in a range from 0 g/L up to 10 g/L resulted in a reduced maximum and overall growth rate µ by 44% and 30%, respectively. To conclude, acetate was identified as a promising target for future process enhancement and strain engineering. Though, the current study demonstrates that the anaerobic cultivation to synthesize surfactin represents a reasonable perspective and feasible alternative to conventional processes.

Wireless bioreactor for anaerobic cultivation of bacteria.

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.

Anaerobic cultivation of waste activated sludge to inoculate solid state anaerobic co-digestion of agricultural wastes: Effects of different cultivated periods.

This study investigated effects of waste activated sludge (WAS) after anaerobic cultivation as inoculum on solid-state anaerobic digestion (SSAD) of agricultural wastes. WAS was anaerobically cultivated for 0, 20 and 50 days and then used as inoculum for co-digestion at substrate/inoculum (S/I) ratios of 2 and 4, respectively. Results showed that treatments inoculated with cultivated WAS exhibited better buffering capability. The highest cumulative methane production (218 L/kg VS) was achieved when inoculating WAS after 50 days of cultivation at the S/I ratio of 2. Fresh WAS without any anaerobic cultivation as inoculum led to digestion instability with significant acidification and limited biogas production, particularly at the S/I ratio of 4. Microbial analysis deciphered that Methanosarcina was the dominant archaea in all treatments and its relative abundance increased with the extension of WAS cultivation time. Hence, WAS after prolonged anaerobic cultivation could be a favorable inoculum for SSAD of agricultural wastes.

Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System.

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes.

Biological diversity of carbon assimilation among isolates of the yeast Dekkera bruxellensis from wine and fuel-ethanol industrial processes.

Dekkera bruxellensis is considered a spoilage yeast in winemaking, brewing and fuel-ethanol production. However, there is growing evidence in the literature of its biotechnological potential. In this work, we surveyed 29 D. bruxellensis isolates from three countries and two different industrial origins (winemaking and fuel-ethanol production) for the metabolization of industrially relevant sugars. The isolates were characterized by the determination of their maximum specific growth rates, and by testing their ability to grow in the presence of 2-deoxy-d-glucose and antimycin A. Great diversity was observed among the isolates, with fuel-ethanol isolates showing overall higher specific growth rates than wine isolates. Preferences for galactose (three wine isolates) and for cellobiose or lactose (some fuel-ethanol isolates) were observed. Fuel-ethanol isolates were less sensitive than wine isolates to glucose catabolite repression (GCR) induction by 2-deoxy-d-glucose. In strictly anaerobic conditions, isolates selected for having high aerobic growth rates were able to ferment glucose, sucrose and cellobiose at fairly high rates without supplementation of casamino acids or yeast extract in the culture medium. The phenotypic diversity found among wine and fuel-ethanol isolates suggests adaptation to these environments. A possible application of some of the GCR-insensitive, fast-growing isolates in industrial processes requiring co-assimilation of different sugars is considered.

Metagenomic, phylogenetic, and functional characterization of predominant endolithic green sulfur bacteria in the coral Isopora palifera.

BACKGROUND: Endolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown. RESULTS: This study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB's abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP. CONCLUSION: We suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons.

Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes.

The community of microorganisms in the mammalian gastrointestinal tract, referred to as the gut microbiota, influences host physiology and immunity. The last decade of microbiome research has provided significant advancements for the field and highlighted the importance of gut microbes to states of both health and disease. Novel molecular techniques have unraveled the tremendous diversity of intestinal symbionts that potentially influence the host, many proof-of-concept studies have demonstrated causative roles of gut microbial communities in various pathologies, and microbiome-based approaches are beginning to be implemented in the clinic for diagnostic purposes or for personalized treatments. However, several challenges for the field remain: purely descriptive reports outnumbering mechanistic studies and slow translation of experimental results obtained in animal models into the clinics. Moreover, there is a dearth of knowledge regarding how gut microbes, including novel species that have yet to be identified, impact host immune responses. The sheer complexity of the gut microbial ecosystem makes it difficult, in part, to fully understand the microbiota-host networks that regulate immunity. In the present manuscript, we review key findings on the interactions between gut microbiota members and the immune system. Because culturing microbes allows performing functional studies, we have emphasized the impact of specific taxa or communities thereof. We also highlight underlying molecular mechanisms and discuss opportunities to implement minimal microbiome-based strategies.

Formate and Nitrate Utilization in Enterobacter aerogenes for Semi-Anaerobic Production of Isobutanol.

Anaerobic bioprocessing is preferred because of its economic advantages. However, low productivity and decreased growth of the host strain have limited the use of the anaerobic process. Anaerobic respiration can be applied to anoxic processing using formate and nitrate metabolism to improve the productivity of value-added metabolites. A isobutanol-producing strains is constructed using Enterobacter aerogenes as a host strain by metabolic engineering approaches. The byproduct pathway (ldhA, budA, and pflB) is knocked out, and heterologous keto-acid decarboxylase (kivD) and alcohol dehydrogenase (adhA) are expressed along with the L-valine synthesis pathway (ilvCD and budB). The pyruvate formate-lyase mutant shows decreased growth rates when cultivated in semi-anaerobic conditions, which results in a decline in productivity. When formate and nitrate are supplied in the culture medium, the growth rates and amount of isobutanol production is restored (4.4 g L-1 , 0.23 g g-1 glucose, 0.18 g L-1 h-1 ). To determine the function of the formate and nitrate coupling reaction system, the mutant strains that could not utilize formate or nitrate is contructed. Decreased growth and productivity are observed in the nitrate reductase (narG) mutant strain. This is the first report of engineering isobutanol-producing E. aerogenes to increase strain fitness via augmentation of formate and nitrate metabolism during anaerobic cultivation.

Effect of water management and silicon on germination, growth, phosphorus and arsenic uptake in rice.

Silicon (Si) is the 2nd most abundant element in soil which is known to enhance stress tolerance in wide variety of crops. Arsenic (As), a toxic metalloid enters into the human food chain through contaminated water and food or feed. To alleviate the deleterious effect of As on human health, it is a need of time to find out an effective strategy to reduce the As accumulation in the food chain. The experiments were conducted during September-December 2014, and 2016 to optimize Si concentration for rice (Oryza sativa L.) exposed to As stress. Further experiment were carried out to evaluate the effect of optimum Si on rice seed germination, seedling growth, phosphorus and As uptake in rice plant. During laboratory experiment, rice seeds were exposed to 150 and 300µM As with and without 3mM Si supplementation. Results revealed that As application, decreased the germination up to 40-50% as compared to control treatment. Arsenic stress also significantly (P < 0.05) reduced the seedling length but Si supplementation enhanced the seedlings length. Maximum seedling length (4.94cm) was recorded for 3mM Si treatment while, minimum seedling length (0.60cm) was observed at day7 by the application of 300µM As. Silicon application resulted in 10% higher seedling length than the control treatment. In soil culture experiment, plants were exposed to same concentrations of As and Si under aerobic and anaerobic conditions. Irrigation water management, significantly (P˂0.05) affected the plant growth, Si and As concentrations in the plant. Arsenic uptake was relatively less under aerobic conditions. The maximum As concentration (9.34 and 27.70mgkg DW-1 in shoot and root, respectively) was found in plant treated with 300µM As in absence of Si under anaerobic condition. Similarly, anaerobic condition resulted in higher As uptake in the plants. The study demonstrated that aerobic cultivation is suitable to decrease the As uptake and in rice exogenous Si supply is beneficial to decrease As uptake under both anaerobic and aerobic conditions.

Cultured microbes represent a substantial fraction of the human and mouse gut microbiota.

During the last 15 years, molecular techniques have been preferred over culture-based approaches for the study of mammalian gut microbiota, i.e. the communities of microorganisms dwelling in the intestine of mammals. The main reason is the belief that the majority of gut bacteria, especially strict anaerobes, escape cultivation. Despite numerous such statements in publications, the literature does not provide a clear overview on the subject. In the present manuscript, we highlight recent work on the cultivation of bacteria from the intestine of mammals, review the literature and provide novel data pertaining to cultured fractions of mammalian gut microbiota. These data show that, despite marked inter-sample variations, 35 to 65% of molecular species detected by sequencing have representative strains in culture.

Method for Indirect Quantification of CH4 Production via H2O Production Using Hydrogenotrophic Methanogens.

Hydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. Methanogens exhibit extraordinary ecological, biochemical, and physiological characteristics and possess a huge biotechnological potential. Yet, the only possibility to assess the methane (CH4) production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH4. In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH4 production potential we developed a novel method for indirect quantification of the volumetric CH4 production rate by measuring the volumetric water production rate. This method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was estimated by determining the difference in mass increase in a quasi-isobaric setting. This novel CH4 quantification method is an accurate and precise analytical technique, which can be used to rapidly screen pure cultures of methanogens regarding their volumetric CH4 evolution rate. It is a cost effective alternative determining CH4 production of methanogens over CH4 quantification by using gas chromatography, especially if applied as a high throughput quantification method. Eventually, the method can be universally applied for quantification of CH4 production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens.

The mouse gut microbiome revisited: From complex diversity to model ecosystems.

Laboratory mice are the most commonly used animal model in translational medical research. In recent years, the impact of the gut microbiota (i.e. communities of microorganisms in the intestine) on host physiology and the onset of diseases, including metabolic and neuronal disorders, cancers, gastrointestinal infections and chronic inflammation, became a focal point of interest. There is abundant evidence that mouse phenotypes in disease models vary greatly between animal facilities or commercial providers, and that this variation is associated with differences in the microbiota. Hence, there is a clear discrepancy between the widespread use of mouse models in research and the patchwork knowledge on the mouse gut microbiome. In the present manuscript, we summarize data pertaining to the diversity and functions of the mouse gut microbiota, review existing work on gnotobiotic mouse models, and discuss challenges and opportunities for current and future research in the field.

First Hungarian case of an infection caused by multidrug-resistant Bacteroides fragilis strain.

We report a case caused by a multidrug-resistant (MDR) Bacteroides fragilis strain isolated from abdominal fluid of a male patient with complex underlying diseases. The patient had received antibiotics prior to the presented case. As far as we know, this case with a MDR B. fragilis is the first from Hungary, and Eastern-Europe, as well.