Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria.

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

Revealing the diversity of bacteria and fungi in the active layer of permafrost at Spitsbergen island (Arctic) - Combining classical microbiology and metabarcoding for ecological and bioprospecting exploration.

Arctic soils are constantly subjected to extreme environmental conditions such as low humidity, strong winds, high salinity, freeze-thaw cycles, UV exposition, and low nutrient availability, therefore, they have developed unique microbial ecosystems. These environments provide excellent opportunities to study microbial ecology and evolution within pristine (i.e. with limited anthropogenic influence) regions since the High Arctic is still considered one of the wildest and least explored environments on the planet. This environment is also of interest for the screening and recovery of unique microbial strains suitable for various biotechnological applications. In this study, a combination of culture-depended and culture-independent approaches was used to determine the cultivation bias in studies of the diversity of cold-active microorganisms. Cultivation bias is a reduction in recovered diversity, introduced when applying a classical culturing technique. Six different soil types, collected in the vicinity of the Polish Polar Station Hornsund (Spitsbergen, Norway), were tested. It was revealed that the used media allowed recovery of only 6.37 % of bacterial and 20 % of fungal genera when compared with a culture-independent approach. Moreover, it was shown that a combination of R2A and Marine Broth media recovered as much as 93.6 % of all cultivable bacterial genera detected in this study. Based on these results, a novel protocol for genome-guided bioprospecting, combining a culture-dependent approach, metabarcoding, next-generation sequencing, and genomic data reuse was developed. With this methodology, 14 psychrotolerant, multi-metal-resistant strains, including the highly promising Rhodococcus spp., were obtained. These strains, besides increased metal tolerance, have a petroleum hydrocarbon utilization capacity, and thus may be good candidates for future bioremediation technologies, also suited to permanently cold regions.

Diversity of antibiotic resistance gene variants at subsequent stages of the wastewater treatment process revealed by a metagenomic analysis of PCR amplicons.

Wastewater treatment plants have been recognised as point sources of various antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) which are considered recently emerging biological contaminants. So far, culture-based and molecular-based methods have been successfully applied to monitor antimicrobial resistance (AMR) in WWTPs. However, the methods applied do not permit the comprehensive identification of the true diversity of ARGs. In this study we applied next-generation sequencing for a metagenomic analysis of PCR amplicons of ARGs from the subsequent stages of the analysed WWTP. The presence of 14 genes conferring resistance to different antibiotic families was screened by PCR. In the next step, three genes were selected for detailed analysis of changes of the profile of ARG variants along the process. A relative abundance of 79 variants was analysed. The highest diversity was revealed in the ermF gene, with 52 variants. The relative abundance of some variants changed along the purification process, and some ARG variants might be present in novel hosts for which they were currently unassigned. Additionally, we identified a pool of novel ARG variants present in the studied WWTP. Overall, the results obtained indicated that the applied method is sufficient for analysing ARG variant diversity.

Draft Genome Sequence of Arctic, Heavy Metal-Resistant Agrococcus sp. Strain ARC_14 Isolated from Active Layer of Permafrost from Spitsbergen (Norway).

Extreme environmental conditions observed in polar regions create a unique ecological niche for microbial life. Bacteria living under these harsh, environmental conditions exhibit specific metabolic capabilities. In this report, we present multimetal-resistant Agrococcus sp. strain ARC_14, isolated from soil samples collected in Spitsbergen, Svalbard, Norway.

Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance (mcr) genes in various environmental settings.

Antibiotic resistance is considered one of the biggest threats to public health and has become a major concern for governments and international organizations. Combating this problem starts with improving global surveillance of antibiotic resistance genes (ARGs) and applying standardized protocols, both in a clinical and environmental context, in agreement with the One Health approach. Exceptional efforts should be directed to controlling ARGs conferring resistance to Critically Important Antimicrobials (CIA). In this study, a systematic literature review to synthesize data on the identification of mcr genes using a PCR technique was performed. Additionally, a novel set of PCR primers for mcr-1 - mcr-9 genes detection was proposed. The developed primers were in silico and experimentally validated by comparison with mcr-specific PCR primers reported in the literature. This validation, besides being a proof-of-concept for primers' usefulness, provided insight into the distribution of mcr genes in municipal wastewater, clay and river sediments, glacier moraine, manure, seagulls and auks feces and daphnids from four countries. This analysis proved that commonly used primers may deliver false results, and some mcr genes may be overlooked in tested samples. Newly-developed PCR primers turned out to be relevant for the screening of mcr genes in various environments.

Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle.

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.

Metaplasmidome-encoded functions of Siberian low-centered polygonal tundra soils.

Plasmids have the potential to transfer genetic traits within bacterial communities and thereby serve as a crucial tool for the rapid adaptation of bacteria in response to changing environmental conditions. Our knowledge of the environmental pool of plasmids (the metaplasmidome) and encoded functions is still limited due to a lack of sufficient extraction methods and tools for identifying and assembling plasmids from metagenomic datasets. Here, we present the first insights into the functional potential of the metaplasmidome of permafrost-affected active-layer soil-an environment with a relatively low biomass and seasonal freeze-thaw cycles that is strongly affected by global warming. The obtained results were compared with plasmid-derived sequences extracted from polar metagenomes. Metaplasmidomes from the Siberian active layer were enriched via cultivation, which resulted in a longer contig length as compared with plasmids that had been directly retrieved from the metagenomes of polar environments. The predicted hosts of plasmids belonged to Moraxellaceae, Pseudomonadaceae, Enterobacteriaceae, Pectobacteriaceae, Burkholderiaceae, and Firmicutes. Analysis of their genetic content revealed the presence of stress-response genes, including antibiotic and metal resistance determinants, as well as genes encoding protectants against the cold.

Simple, Reliable, and Time-Efficient Manual Annotation of Bacterial Genomes with MAISEN.

Over the last 15 years, the costs of DNA sequencing have sharply fallen, effectively shifting the costs of DNA analysis from sequencing to bioinformatic curation and storage. A huge number of available DNA sequences (including genomes and metagenomes) resulted in the development of various tools for sequence annotation. While much effort has been invested into the development of automatic annotation pipelines, manual curation of their results is still necessary in order to obtain a reliable and strictly validated data. Unfortunately, due to its time-consuming nature, manual annotation is now rarely used.In this chapter, a protocol for efficient manual annotation of prokaryotic DNA sequences using a novel bioinformatic tool-MAISEN ( http://maisen.ddlemb.com ), is presented. MAISEN is a free, web-based tool designed to accelerate manual annotation, by providing the user with simple interface and precomputed alignments for each predicted feature. It was designed to be available for every scientist, regardless of their bioinformatic proficiency.

PbS biomineralization using cysteine: Bacillus cereus and the sulfur rush.

Bacillus sp. Abq, belonging to Bacillus cereus sensu lato, was isolated from an aquifer in New Mexico, USA and phylogenetically classified. The isolate possesses the unusual property of precipitating Pb(II) by using cysteine, which is degraded intracellularly to hydrogen sulfide (H2S). H2S is then exported to the extracellular environment to react with Pb(II), yielding PbS (galena). Biochemical and growth tests showed that other sulfur sources tested (sulfate, thiosulfate, and methionine) were not reduced to hydrogen sulfide. Using equimolar concentration of cysteine, 1 mM of soluble Pb(II) was removed from Lysogeny Broth (LB) medium within 120 h of aerobic incubation forming black, solid PbS, with a removal rate of 2.03 µg L-1 h-1 (∼8.7 µM L-1 h-1). The mineralogy of biogenic PbS was characterized and confirmed by XRD, HRTEM and EDX. Electron microscopy and electron diffraction identified crystalline PbS nanoparticles with a diameter <10 nm,  localized in the extracellular matrix and on the surface of the cells. This is the first study demonstrating the use of cysteine in Pb(II) precipitation as insoluble PbS and it may pave the way to PbS recovery from secondary resources, such as Pb-laden industrial effluents.

Genome-Wide and Functional View of Proteolytic and Lipolytic Bacteria for Efficient Biogas Production through Enhanced Sewage Sludge Hydrolysis.

In this study, we used a multifaceted approach to select robust bioaugmentation candidates for enhancing biogas production and to demonstrate the usefulness of a genome-centric approach for strain selection for specific bioaugmentation purposes. We also investigated the influence of the isolation source of bacterial strains on their metabolic potential and their efficiency in enhancing anaerobic digestion. Whole genome sequencing, metabolic pathway reconstruction, and physiological analyses, including phenomics, of phylogenetically diverse strains, Rummeliibacillus sp. POC4, Ochrobactrum sp. POC9 (both isolated from sewage sludge) and Brevundimonas sp. LPMIX5 (isolated from an agricultural biogas plant) showed their diverse enzymatic activities, metabolic versatility and ability to survive under varied growth conditions. All tested strains display proteolytic, lipolytic, cellulolytic, amylolytic, and xylanolytic activities and are able to utilize a wide array of single carbon and energy sources, as well as more complex industrial by-products, such as dairy waste and molasses. The specific enzymatic activity expressed by the three strains studied was related to the type of substrate present in the original isolation source. Bioaugmentation with sewage sludge isolates-POC4 and POC9-was more effective for enhancing biogas production from sewage sludge (22% and 28%, respectively) than an approach based on LPMIX5 strain (biogas production boosted by 7%) that had been isolated from an agricultural biogas plant, where other type of substrate is used.

Literature-based, manually-curated database of PCR primers for the detection of antibiotic resistance genes in various environments.

A literature-based, manually-curated database of PCR primers for the detection of antibiotic resistance genes in various environments was constructed (LCPDb-ARG; lcpdb.ddg.biol.uw.edu.pl and lcpdb.ddlemb.com). Currently, this database is comprised of 607 PCR primer pairs designed for the amplification of various genes conferring resistance to antibiotics representing 10 classes of antimicrobial agents. Three parameters were assigned for each primer pair: specificity, efficacy and taxonomic efficacy. These parameters were evaluated using a novel bioinformatic tool, UniPriVal, developed for this study. UniPriVal was used to validate each primer pair against various databases, including the Bacterial Antimicrobial Resistance Reference Gene Database (BARRGDB) and those of the National Center of Biotechnology Information (NCBI). Primer pairs specific for each antibiotic resistance gene were ranked based on their model success metric value. To validate the utility and correctness of the information collected in the LCPDb-ARG, selected primer pairs were tested in bioinformatic and experimental PCR surveys. To our knowledge, this is the first database designed to facilitate PCR monitoring of the occurrence and diversity of antibiotic resistance genes in environmental and clinical samples. The internal validation system of this user-friendly application enables the quantified ranking of PCR primer pairs, which assists selection of the best primers for each application.

Genomic and Biotechnological Characterization of the Heavy-Metal Resistant, Arsenic-Oxidizing Bacterium Ensifer sp. M14.

Ensifer (Sinorhizobium) sp. M14 is an efficient arsenic-oxidizing bacterium (AOB) that displays high resistance to numerous metals and various stressors. Here, we report the draft genome sequence and genome-guided characterization of Ensifer sp. M14, and we describe a pilot-scale installation applying the M14 strain for remediation of arsenic-contaminated waters. The M14 genome contains 6874 protein coding sequences, including hundreds not found in related strains. Nearly all unique genes that are associated with metal resistance and arsenic oxidation are localized within the pSinA and pSinB megaplasmids. Comparative genomics revealed that multiple copies of high-affinity phosphate transport systems are common in AOBs, possibly as an As-resistance mechanism. Genome and antibiotic sensitivity analyses further suggested that the use of Ensifer sp. M14 in biotechnology does not pose serious biosafety risks. Therefore, a novel two-stage installation for remediation of arsenic-contaminated waters was developed. It consists of a microbiological module, where M14 oxidizes As(III) to As(V) ion, followed by an adsorption module for As(V) removal using granulated bog iron ores. During a 40-day pilot-scale test in an abandoned gold mine in Zloty Stok (Poland), water leaving the microbiological module generally contained trace amounts of As(III), and dramatic decreases in total arsenic concentrations were observed after passage through the adsorption module. These results demonstrate the usefulness of Ensifer sp. M14 in arsenic removal performed in environmental settings.

Genome-Guided Characterization of Ochrobactrum sp. POC9 Enhancing Sewage Sludge Utilization-Biotechnological Potential and Biosafety Considerations.

Sewage sludge is an abundant source of microorganisms that are metabolically active against numerous contaminants, and thus possibly useful in environmental biotechnologies. However, amongst the sewage sludge isolates, pathogenic bacteria can potentially be found, and such isolates should therefore be carefully tested before their application. A novel bacterial strain, Ochrobactrum sp. POC9, was isolated from a sewage sludge sample collected from a wastewater treatment plant. The strain exhibited lipolytic, proteolytic, cellulolytic, and amylolytic activities, which supports its application in biodegradation of complex organic compounds. We demonstrated that bioaugmentation with this strain substantially improved the overall biogas production and methane content during anaerobic digestion of sewage sludge. The POC9 genome content analysis provided a deeper insight into the biotechnological potential of this bacterium and revealed that it is a metalotolerant and a biofilm-producing strain capable of utilizing various toxic compounds. The strain is resistant to rifampicin, chloramphenicol and ß-lactams. The corresponding antibiotic resistance genes (including blaOCH and cmlA/floR) were identified in the POC9 genome. Nevertheless, as only few genes in the POC9 genome might be linked to pathogenicity, and none of those genes is a critical virulence factor found in severe pathogens, the strain appears safe for application in environmental biotechnologies.

The effect of high hydrostatic pressure treatment on the molecular structure of starches with different amylose content.

The effect of high hydrostatic pressure processing (650MPa/9min) on molecular mass distribution, and hydrodynamic and structural parameters of amylose (maize, sorghum, Hylon VII) and amylopectin (waxy maize, amaranth) starches was studied. The starches were characterized by high-performance size-exclusion chromatography (HPSEC) equipped with static light scattering and refractive index detectors and by Fourier Transform Infrared (FTIR) spectroscopy. Significant changes were observed in molecular mass distribution of pressurized waxy maize starch. Changes in branches/branch frequency, intrinsic viscosity, and radius of gyration were observed for all treated starches. The combination of SEC and FTIR data showed that α-1,6-glycosidic bonds are more frequently split in pressurized amaranth, Hylon VII, and waxy maize starch, while in sorghum and maize starches, the α-1,4 bonds are most commonly split. Our results show that the structural changes found for pressurized starches were more strongly determined by the starch origin than by the processing applied.

Molecular characterization of the pA3J1 plasmid from the psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_J3.

The knowledge on plasmids of cold-active bacteria is highly limited. In this study, the molecular characterization of the pA3J1 plasmid of Antarctic psychrotolerant bacterium Pseudomonas sp. ANT_J3 was performed. Within this plasmid, thirteen putative open reading frames were identified. Nine of them encoded proteins involved in replication, partitioning, postsegregational elimination of plasmid-less cells (via a toxin-antitoxin system activity), multimer resolution and mobilization by conjugal transfer. These genes constitute the plasmid backbone. The functional analysis of the pA3J1 maintenance region revealed that it is a narrow host range replicon, stably maintained in the host cells by the combined activities of the partitioning and relBE-type toxin-antitoxin systems. It was also suggested that the replication system of the pA3J1 plasmid may be temperature-sensitive. Comparative analyses revealed the presence of 16 Pseudomonas plasmids encoding homologous replication proteins and 5 plasmids carrying mobA genes homologous to the corresponding gene of pA3J1. The relaxase (MobA) of the pA3J1 plasmid was classified into MOBQ family, and the phylogenetic analysis suggested that this may be a representative of a novel group (or subgroup) within this family. The structural and comparative analyses revealed that the arrangement of genetic modules in the pA3J1 plasmid is unique.

In vitro characteristics of Lactobacillus spp. strains isolated from the chicken digestive tract and their role in the inhibition of Campylobacter colonization.

Campylobacter jejuni/coli infections are the leading cause of bacterial diarrheal illnesses in humans. Many epidemiological studies indicate that improperly prepared meat from chickens that carry a high load of Campylobacter in their intestinal tracts is the key source of human infections. LAB, mainly members of the Lactococcus and Lactobacillus genera, increasingly have been tested as vehicles for the delivery of heterologous bacterial or viral antigens to animal mucosal immune systems. Thus, the objective of this study was to isolate, identify, and characterize Lactobacillus spp. strains isolated from chickens bred in Poland. Their ability to decrease the level of bird gut colonization by C. jejuni strain was also analyzed. First, the influence of the different chicken rearing systems was evaluated, especially the effect of diets on the Lactobacillus species that colonize the gut of chickens. Next, selected strains were analyzed in terms of their anti-Campylobacter activity in vitro; potential probiotic traits such as adhesion properties, bile and low pH tolerance; and their ability to grow on a defined carbon source. Given that improperly prepared chicken meat is the main source of human infection by Campylobacter, the selected strains were also assessed for their ability to inhibit Campylobacter colonization in the bird's intestine. These experiments revealed enormous physiological diversity among the Lactobacillus genus strains. Altogether, our results showed that L. plantarum strains isolated from the digestive tracts of chickens bred in Poland displayed some probiotic attributes in vitro and were able to decrease the level of bird gut colonization by C. jejuni strain. This suggests that they can be employed as vectors to deliver Campylobacter immunodominant proteins to the bird's immune system to strengthen the efficacy of in ovo vaccination.

A preliminary study about the influence of high hydrostatic pressure processing in parallel with oak chip maceration on the physicochemical and sensory properties of a young red wine.

The influence of high hydrostatic pressure (HHP) processing in parallel with oak chip maceration on the physicochemical and sensory properties of a young red wine was investigated preliminarily. Wines were treated by HHP at 250, 450 and 650MPa for up to 45min and French oak chips (5g/L) were added. HHP enhanced the extraction of phenolics from oak chips. The phenolic contents and antioxidant activity of the wine increased after HHP processing in the presence of oak chips. Meanwhile, the anthocyanin content and wine color intensity decreased in the first 5min of pressure treatment and then increased gradually. The multivariate analysis revealed that "pressure holding time" was the key factor affecting wine physicochemical characteristics during HHP processing in the presence of oak chips. Furthermore, oak chip maceration with and without HHP processing weakened the intensities of several sensory attributes and provided the wine with an artificial taste.

In vitro release of theophylline from starch-based matrices prepared via high hydrostatic pressure treatment and autoclaving.

Recent works have demonstrated that release behavior of bioactive compounds varies with the nature of the matrix regarding its chemical composition, morphology and surface properties. Starch matrices varying in amylose content (maize, sorghum, Hylon VII) or pure amylopectin ones (waxy maize, amaranth starch), containing theophylline (10 mg, 50 mg/0.5 g of starch), were obtained via high hydrostatic pressure treatment (650 MPa/9 min) and autoclaving (120 °C/20 min). Both the treatment used and drug dose affected the theophylline release profiles from the matrices studied. The profiles of amylopectin starch matrices satisfactorily fitted with selected mathematical models, indicating a controlled theophylline release. The principal component analysis confirmed substantial differences in drug release between the amylose and amylopectin matrices. The differences in matrix morphology, internal surface area and porosity (mesopore diameter, cumulative pore volume) between the matrices studied were found to be key factors affecting the theophylline dissolution.