Bacterial strains were isolated from different locations and screened for plant growth promoting (PGP) features. Comparative analyses of the 16S gene sequences of 5 of the strains indicated taxonomic relatedness within the genus Paenibacillus. A polyphasic taxonomic approach was employed to study the strains in detail to clarify their phylogenetic position. Genome-based analyses, including digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI), revealed values consistently below the accepted species delineation thresholds when compared with closest relatives of each strain. Additionally, the strains showed clear differences in their physiological and biochemical profiles to the type strains of the closest related species. A notably diverse set of genes potentially involved in plant growth promotion was detected in all strains. With respect to the analyses reported here, the following new names are proposed: Paenibacillus corni sp. nov., with AK-167T as the type strain (= LMG 34403T = DSM 121689T); Paenibacillus vaccinii sp. nov., with AK-264T as the type strain (= LMG 34404T = DSM 121714T); Paenibacillus ericacearum sp. nov., with AK-265T as the type strain (= CCM 6913T = LMG 34405T = DSM 121715T); Paenibacillus polytrichii sp. nov., with AK-286T as the type strain (= CCM 9614T = LMG 34406T = DSM 121716T); and Paenibacillus artemisiae sp. nov., with DT-106T as the type strain (= LMG 34409T = CCM 9610T = DSM 121690T).

Several bacterial strains affiliated with the genera Pseudomonas and Zestomonas were isolated from rhizosphere samples and screened for plant growth-promoting (PGP) traits. Nine strains were analyzed polyphasically, showing ANI and dDDH values below or near species thresholds, together with phenotypic and biochemical traits supporting their differentiation. Genomic analyses revealed a repertoire of PGP-associated functions, including vitamin and cofactor biosynthesis (riboflavin, cobalamin, and thiamin), and high-affinity nutrient acquisition systems as phosphate transporters and phosphonate utilization. All strains encoded traits relevant to the rhizosphere inferred from genome annotation, including genes involved in auxin and cytokinin biosynthesis, oxidative stress tolerance, dissimilatory nitrate reduction, sulfur assimilation, and siderophore production. Metagenomic screening showed that most species are globally distributed across plant-associated, soil, freshwater, and animal-associated habitats. Based on phylogenetic, genomic, and phenotypic evidence, the strains represent seven novel species: Pseudomonas rosaeacicularis sp. nov., with AK-381T as the type strain (= LMG 34445T = CCM 9596T); Pseudomonas corni sp. nov., with AK-10T as the type strain (= CCM 9599T = LMG 34325T); Pseudomonas oplopanacis sp. nov., with AK-188T as the type strain (= CCM 9593T = LMG 34326T); Pseudomonas salicis sp. nov., with AK-309T as the type strain (= CCM 9595T = LMG 34328T); Pseudomonas artemisiae sp. nov., with DT-100T as the type strain (= LMG 32880T = DSM 115114T = CCM 9281T); Pseudomonas imperatae sp. nov., with ST-212T as the type strain (CCM 9594T = LMG 34330T); and Zestomonas ipomoeae sp. nov., with ST-55T as the type strain (LMG 32881T = CCM 9283T = DSM 115239T).

Climate change and an increasing global population are supposed to cause progressive water shortages in the future. Using rainwater in water tanks for evaporative cooling systems as a substitute might help reduce water consumption needed for cooling. Therefore, our study identified the present microbiota in evaporative cooling systems (ECS) and used an ex situ approach to identify effects of the substitution of tap water with rainwater. Culturable cell counts (CCC) based on colony-forming units for total counts or selected pathogens, total biofilm masses based on crystal violet staining, and the bacterial composition (16S rRNA sequencing) of biofilms sampled in different ECS were considered. Data suggest that rainwater does not enhance biofilm growth, since both culturable cells and total biofilm mass tended to decrease when using rainwater instead of tap water. Interestingly, biocide treatment slightly enhanced the total biofilm mass and CCC. Sequencing data revealed differences in the microbiota when using the different water types; yet, the community shift did not include an enrichment of typical pathogens in both cases. Overall, our results suggest that a substitution of tap water with rainwater for ECS creates no disadvantage with regard to the microbial safety and might allow a reduction of financial and resource investments.

Two novel bacterial strains, designated MP11MiT and MP213FoT, were isolated from the burying beetle Nicrophorus vespilloides and subjected to a polyphasic taxonomic study. Strain MP11MiT was placed in the genus Gordonia, showing the highest 16S rRNA gene sequence similarities to Gordonia liuliyuniae HY366T (98.8% sequence identity), followed by Gordonia zhenghanii HY002T (98.4%). The strain exhibited low genomic relatedness to its closest relatives [ANI (average nucleotide identity) <90% and dDDH (digital DNA-DNA hybridization) <37%], far below the accepted species thresholds (95-96% ANI and 70% dDDH), unambiguously supporting species-level distinctiveness. Strain MP213FoT was affiliated with the genus Pseudochrobactrum, with Pseudochrobactrum algeriense LMG 32378T as its closest relative. ANI (79.4-79.9%), dDDH (22.8-23.1%), average amino acid identity (82.5-83.8%) and percentage of conserved proteins (71.6-77.2%) values confirmed its clear separation from all hitherto recognized species of this genus. The genome of MP213FoT comprises a circular chromosome and a plasmid carrying conserved genus-specific features. Analysis of specialized biosynthetic potential identified 13 gene clusters in MP11MiT and 4 in MP213FoT, highlighting their capacity for specialized metabolite biosynthesis. Based on phylogenomic, chemotaxonomic and phenotypic characteristics, strains MP11MiT and MP213FoT are proposed as representatives of two novel species, for which the names Gordonia abscondita sp. nov. (type strain MP11MiT=DSM 113667=CCM 9219) and Pseudochrobactrum mosebachae sp. nov. (type strain MP213FoT=DSM 113682=CCM 9218) are introduced.

Two motile bacterial strains, designated as cfHf56-1 T and SW 252, were isolated from the coelomic fluid of Holothuria forskali and from the surrounding seawater at the animal sampling site, respectively. The sea cucumber was collected in the Glénan archipelago (Brittany, France). Strains cfHf56-1 T and SW 252 were Gram-stain-negative, non-spore-forming and rod-shaped bacteria. Colonies made on marine agar plates were brown in color. The pH and temperature ranges for growth were 7–8 and 18–30 °C, respectively, in marine broth. The major fatty acids were 16 : 1 cis9 and 16 : 0. Phylogenetic analyses evidenced that both strains belong to a novel species in the genus Pseudoalteromonas . The strains were closely related to the type strains of Pseudoalteromonas caenipelagi , Pseudoalteromonas byunsanensis and Pseudoalteromonas amylolytica, with 75–78 % ANI and 19–21 % dDDH values. In this context, cfHf56-1 T (= CIP 111854 T = CECT 30642 T ) is considered as the type strain of the novel species for which the name Pseudoalteromonas holothuriae sp. nov. is proposed. The genome of the type strain is characterized by a size of 5.1 Mbp and a G+C content of 40.5%.

Bacteria belonging to the genera Chryseobacterium, Sphingomonas, Variovorax, Neorhizobium, Parapedobacter and Erwinia have been isolated from different soils and have also been shown to exhibit plant growth promoting (PGP) features. A polyphasic approach was employed to ascertain the taxonomic status of eight selected strains all isolated from dry soils in association with plants showing high desiccation tolerances. Based on the result of the polyphasic characterization, the following names are proposed: Chryseobacterium hilariae sp. nov., with DT-3T as the type strain (= CIP 112171T = LMG 32722T = CCM 9258T); Sphingomonas astragali sp. nov., with DT-204T as the type strain (= CCM 9255T = LMG 33194T = CIP 112175T); Sphingomonas radicis sp. nov., with DT-207T as the type strain (= CCM 9257T = LMG 32727T = DSM 114514T); Sphingomonas larreae sp. nov., with DT-51T as the type strain (= CCM 9259T = CIP 112177T = DSM 114511T = LMG 32723T); Variovorax stachyos sp. nov., with DT-64T as the type strain (= LMG 32724T = DSM 114531T = CIP 112170T); Erwinia artemisiae sp. nov., with DT-104T as the type strain (CCM 9256T = LMG 32725T = DSM 114512T); Parapedobacter brassicae sp. nov., with DT-150T as the type strain (LMG 32759T = DSM 115120T); and Neorhizobium descurainiae sp. nov., with DT-125T as the type strain (LMG 32760T = CIP 112184T).

AIMS:Lactic acid bacteria (LAB) are crucial for the quality and preservation of silage. Exploration and isolation of the key microbiome are important for improving fermentation processes. While cultivation-dependent methods enable the isolation of LAB, they often overlook organisms with challenging cultivation requirements. Next-generation sequencing provides cultivation-independent microbiome insights. In contrast to commonly used methods, long-read sequencing platforms like the Pacific Biosciences (PacBio) single-molecule real-time (SMRT) platform offer species-level resolution. This study aims to investigate the benefits of a combined cultivation-dependent and -independent approach for silage microbiome analyses. METHODS AND RESULTS:The common isolation medium for LAB was supplemented with sterile pressed juice from plant material improving the cultivation conditions. PacBio amplicon sequencing provided an almost complete and cultivation-independent picture of the bacterial community. The use of mock communities and a live/dead discriminating treatment of the samples revealed that the analysis can be misleading if appropriate controls are not performed. CONCLUSION:Growth of plant-associated microorganisms can be supported by a plant juice containing isolation medium, allowing isolation of the dominant LAB from silage. Microbial dynamics can be represented using long-read sequencing. However, the use of controls such as a live/dead discrimination and mock communities is essential for the reliability of the data.

Members of the genera Flavobacterium, Chryseobacterium and Sphingomonas constitute a group of microorganisms in the rhizosphere associated with plant growth promoting (PGP) features. A polyphasic approach was employed to ascertain the taxonomic status of five selected strains. Overall genome relatedness indices of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) between the strains and the other members of the genera Flavobacterium, Chryseobacterium and Sphingomonas were found to be below the established thresholds, respectively. Morphological, physiological, and biochemical characteristics of the strains confirmed their status as five novel species. A large variety of genes involved in plant growth promotion and carbohydrate utilization were found in all strains suggesting a contribution of all strains to PGP. Based on the result of the polyphasic characterization, the following names are proposed: Chryseobacterium terrae sp. nov., with the strain ST-37T as the type strain (= CCM 9260T = LMG 32728T); Flavobacterium plantiphilum sp. nov., with the strain ST-87T as the type strain CIP 112180T = DSM 114790T = LMG 32757T); Flavobacterium rhizophilum sp. nov., with the strain ST-75T as the type strain (= CIP 112185T = DSM 114831T = LMG 32758T); Flavobacterium rhizosphaerae sp. nov., with the strain ST-119T as the type strain (CIP 112181T = DSM 114832T = LMG 32756T); and Sphingomonas plantiphila sp. nov. with the strain ST-64 T as the type strain (= CCM 9261T = CIP 112178T = DSM 114515T = LMG 32729T).

Abstract Background Long-read 16S rRNA gene amplicon sequencing has a high potential for characterizing food-associated microbiomes. The advantage results from sequencing the full-length (1,500 bp) gene, enabling taxonomic resolution at species level. Here we present a benchmarking study using mock communities representative of milking machine biofilms and raw meat, revealing challenges relevant to food-associated habitats. These were varying species abundances, reliable intra-genus differentiation of species, and detection of novel species with < 98.7% sequence identity to type strains. By using mock communities at different levels of preparation − as mixed whole cells, mixed extracted DNA, and mixed PCR products − we systematically investigated the influence of DNA extraction using two different kits, PCR amplification of 16S rRNA genes, sequencing, and bioinformatics analysis including reference database and gene copy number normalization on bacterial composition and alpha diversity. Results We demonstrated that PacBio ccs-reads allowed for correct taxonomic assignment of all species present within the mock communities using a custom Refseq database. However, choice of percent identity values for taxonomic assignment had a strong influence on identification and processing of reads from novel species. PCR amplification of 16S rRNA genes produced the strongest bias on the observed community composition, while sequencing alone reproduced the preset composition well. The PCR bias can in part be attributed to differences in mol% G + C content of 16S rRNA genes resulting in preferred amplification of low mol% G + C-containing taxa. Conclusions This study underlines the importance of benchmarking studies with mock communities representing the habitat of interest to evaluate the methodology prior to analyzing real samples of unknown composition. It demonstrates the advantage of long-read sequencing over short-read sequencing, as species level identification enables in-depth characterization of the habitat. One benefit is improved risk assessment by enabling differentiation between pathogenic and apathogenic species of the same genus.

Backgound. As biofilms are known to harbour (multi-)resistant species, their presence in health settings must be considered critical. Although there is evidence that bacteria spread from drains to the outside, there is still a lack of research data focusing on drain biofilms from hospitals. Methods. We sampled biofilms from various wards of Helios Hospital Krefeld (Germany), where comprehensive antibiotic consumption data were available. Biofilms were analysed by cell counting, isolation of relevant bacterial groups and genetic and phenotypical resistance parameters. Data were correlated with the prescribed antibiotics of the respective ward. Furthermore, an ex situ biofilm model was employed to investigate the influence of sub-inhibitory antibiotics on the bacterial community and the prevalence of class 1 integrons. Results. Our results show that every ward harboured medically relevant bacterial species. While no significant differences were found in cell counts, the median prevalence of the resistance marker gene intI1 correlated with the amount of prescribed antibiotics. In contrast, phenotypical resistances showed no similar tendency. In addition, melting curve analysis data and changes in intI1 prevalence show that the composition of the bacterial community shifted depending on the biofilm and antibiotic. Conclusions. To the best of our knowledge, our study is the first considering possible correlations between the consumption data of hospital wards and resistances in drain biofilms the way we did. Based on our results, we conclude that sub-inhibitory concentrations of antibiotics have no general effect on biofilms in terms of bacterial community shift and occurrence of antibiotic-resistant species. Amongst other things, the effect depends on the initial composition of the bacterial community, the antibiotic used and the intrinsic bacterial resistance, e.g., prevalence of class 1 integrons.

The bacterial strains Brochothrix thermosphacta DH-B18 and Rathayibacter sp. DH-RSZ4 were isolated from raw sausage and escalope samples and grown in a CO2-rich modified atmosphere. Here, we present both circular genomes obtained by nanopore sequencing.

The relative abundance of antibiotic-resistant bacteria and antibiotic-resistance genes was surveyed for different parts of a milking machine. A cultivation approach based on swab samples showed a highly diverse microbiota, harboring resistances against cloxacillin, ampicillin, penicillin, and tetracycline. This approach demonstrated a substantial cloxacillin resistance of numerous taxa within milking machine microbiota coming along with regular use of cloxacillin for dry-off therapy of dairy cows. For the less abundant tetracycline-resistant bacteria we found a positive correlation between microbial cell density and relative abundance of tetracycline-resistant microorganisms (R2 = 0.73). This indicated an accelerated dispersion of resistant cells for sampling locations with high cell density. However, the direct quantification of the tetM gene from the swap samples by qPCR showed the reverse relation to bacterial density if normalized against the abundance of 16S rRNA genes (R2 = 0.88). The abundance of 16S rRNA genes was analyzed by qPCR combined with a propidium monoazide treatment, which eliminates 16S rRNA gene signals in negative controls.

Biofilms are the most common growth types of microorganisms. These complex communities usually consist of different species and are embedded in an extracellular matrix containing polymers, proteins and DNA. This matrix offers protection against different (a)biotic environmental factors and generally increases resistances. Higher resistances against antibiotics are one of the main reasons why biofilms are often associated with healthcare settings. Nevertheless, they are also found in domestic settings, mostly in humid places with abundant nutrients like dishwashers or washing machines. Biofilms in these areas show individual compositions and are influenced for example by temperature, frequency of use or the age of the device. In this study, we introduce a model for the ex-situ cultivation of domestic biofilms from household appliances. Furthermore, we tested the ability of high resolution melting analysis (HRMA) as a tool for analysing these biofilms. Our goal was to maintain a high amount of complexity in the ex-situ biofilms that is characterized by the melting behavior of the contained DNA. Dishwasher and washing machine biofilms were sampled in private households and cultivated for 10 d. After DNA extraction, 16S rDNA was sequenced and melting behavior of the bacterial Internal Transcribed Spacer (ITS) region was analysed. Additionally, testing for independence of continuous new sampling, storage of cultivated biofilms in glycerol stocks and following recultivation of them was done up to three times. Our results show that a high level of complexity could be maintained in the ex-situ biofilms after 10 d of cultivation, although in general the bacterial diversity slightly decreased compared to the original biofilm in most cases. Recultivation of a similar biofilm from glycerol stocks was possible as well with some impact by various factors. Differences in the bacterial composition of biofilms could clearly made visible by HRMA although it was not possible to match peaks to a specific phylogenetic group. Still, HRMA proved to be a less costly and time consuming alternative to sequencing for the characterization of biofilms.

High levels of carbon dioxide are known to inhibit the growth of microorganisms. A total of twenty strains of filamentous fungi and yeasts were isolated from habitats with enriched carbon dioxide concentration. Most strains were derived from modified atmosphere packed (MAP) food products or mofettes and were cultivated under an atmosphere of 20% CO2 and 80% O2. The influence of CO2 on fungal cell membrane fatty acid profiles was examined in this study. Major changes were the increase in linolenic acid (C18:3 cis 9, 12, 15) and, additionally in most strains, linoleic acid (C18:2 cis 9, 12) with a maximum of 24.8%, at the expense of oleic (C18:1 cis 9), palmitic (C16:0), palmitoleic (C16:1 cis 9) and stearic acid (C18:0). The degree of fatty acid unsaturation increased for all of the strains in the study, which consequently led to lower melting temperatures of the cell membranes after incubation with elevated levels of CO2, indicating fluidization of the membrane and a potential membrane malfunction. Growth was reduced in 18 out of 20 strains in laboratory experiments and a change in pigmentation was observed in several strains. Two of the isolated strains, strain WT5 and strain WR1, were found to represent a hitherto undescribed yeast for which the new genus and species Stenotrophomyces fumitolerans (MB# 849906) is proposed.

Four Gram-positive, rod-shaped, none-sporeforming, non-motile isolates were obtained from various raw milk samples taken from the cooling tank on a research farm in Königswinter, Germany. Based on phylogenetic analysis of the 16S rRNA genes and whole genome sequences, all isolates were assigned to the genus Corynebacterium, but were divided in two different groups. All isolates contained C18 : 1 cis 9 and C16 : 0 as predominant fatty acids, as well as traces of C18 : 0. They all contained menaquinones MK-8 (H2) and MK-9 (H2) and produced mycolic acids characteristic for the majority of species belonging to the genus Corynebacterium. 16S rRNA gene sequence similarity values to the closest related type strains Corynebacterium humireducens DSM 45392T and Corynebacterium pilosum DSM 20521T were below 98.7 %, average nucleotide identity values were below 86 % and digital DNA-DNA-hybridization values were below 25 %, indicating that the isolates represent two novel species. The names Corynebacterium suedekumii sp. nov. and Corynebacterium breve sp. nov. are proposed, represented by the type strains LM112T (=DSM 116216T=HAMBI 3782T) and R4T (=DSM 116183T=HAMBI 3785T), respectively.

The effects of naringenin and the biflavonoids amentoflavone and tetrahydroamentoflavone on select bacterial lipids (carotenoids, fatty acids, and menaquinones) and membrane fluidity based on Laurdan generalized polarization were investigated. For this purpose, the pigment-forming food-associated microorganisms Staphylococcus xylosus (DSM 20266T and J70), Staphylococcus carnosus DSM 20501T, and Micrococcus luteus (ATCC 9341 and J3) were studied. The results suggest an envelope stress response by microorganisms due to flavonoids and an employment of adaptive mechanisms using carotenoids, fatty acids, and menaquinones. The flavonoid monomer naringenin impacted carotenoids, fatty acids, menaquinones, and membrane fluidity. Naringenin significantly influenced the carotenoid profile, particularly by an increase in the relative proportion of 4,4'-diaponeurosporenoic acid in Staphylococcus xylosus. Amentoflavone caused changes mainly in the membrane of Micrococcus luteus and decreased the menaquinone content. Tetrahydroamentoflavone mainly affected the carotenoids in the investigated strains. The noticeably different CCS value of tetrahydroamentoflavone compared to naringenin and amentoflavone revealed further insights into the structure-dependent effects of flavonoids. This study provides valuable insights into the response of pigment-forming food-associated microorganisms to naringenin, amentoflavone, and tetrahydroamentoflavone, which is important for the targeted and safe application of the latter as natural preservatives and useful for further research on the mechanisms of action.

A Gram-strain positive, aerobic, endospore-forming bacterial strain (JJ-246T) was isolated from the rhizosphere of Zea mays. The 16S rRNA gene sequence similarity comparisons showed a most closely relationship to Paenibacillus oenotherae DT7-4T (98.4%) and Paenibacillus xanthinolyticus 11N27T (98.0%). The pairwise average nucleotide identity and digital DNA-DNA hybridisation values of the JJ-246T genome assembly against publicly available Paenibacillus type strain genomes were below 82% and 33%, respectively. The draft genome of JJ-246T shared many putative plant-beneficial functions contributing (PBFC) genes, related to plant root colonisation, oxidative stress protection, degradation of aromatic compounds, plant growth-promoting traits, disease resistance, drug and heavy metal resistance, and nutrient acquisition. The quinone system of strain JJ-246T, the polar lipid profile and the major fatty acids were congruent with those reported for members of the genus Paenibacillus. JJ-246T was shown to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus plantiphilus sp. nov. is proposed, with JJ-246T (= LMG 32093T = CCM 9089T = CIP 111893T) as the type strain.

The genus Nitrospira represents the dominant nitrite-oxidizing clade in most wastewater treatment plants (WWTPs) globally, and several Nitrospira strains have been isolated from activated sludge. Using a pre-enrichment strategy with alternating nitrifying and denitrifying conditions, followed by incubation at elevated temperatures, we isolated a novel Nitrospira species, named Nitrospira tepida. This moderately thermophilic species with optimal growth between 37 and 45°C is only distantly related to other Nitrospira and forms a novel lineage VII within the genus, together with few environmental 16S rRNA gene sequences predominantly detected in thermal wastewater or oxygen-limited systems. Genomic and physiological analyses revealed remarkable differences between N. tepida and two other isolates previously obtained from the same WWTP, suggesting niche differentiation between these nitrite oxidizers. N. tepida grows in aggregates, and tolerates nitrite and nitrate concentrations of up to 20 mM and 40 mM, respectively. The Km value for nitrite of N. tepida is 77 ± 26 µM. In summary, this novel Nitrospira lineage seems to be well-adapted for wastewater treatment processes at elevated temperatures and limited aeration, conditions that potentially reduce operational costs of such systems.

Two Gram-stain-positive, aerobic, endospore-forming bacterial strains, isolated from the rhizosphere of Zea mays were studied for their detailed taxonomic allocation. Based on 16S rRNA gene sequence similarity comparisons, both strains JJ-7T and JJ-60T were shown to be members of the genus Paenibacillus. Strain JJ-7T was most closely related to the type strains of Paenibacillus tianjinensis (99.6 %) and P. typhae (98.7 %), and strain JJ-60T to Paenibacillus etheri (99.5 %). The 16S rRNA gene sequence similarities to all other Paenibacillus species were ≤98.4 %. Both strains JJ-7T and JJ-60T showed 97.6 % 16S rRNA gene sequence similarity between each other. Genomic comparisons showed that the average nucleotide identity and digital DNA-DNA hybridization values to next related type strain genomes were always <94 and <56 %, respectively. The polar lipid profiles of both strains contain a number of phospholipids including diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, which is in accord with the genus Paenibacillus. The major quinone was MK-7 in both strains. Major fatty acids were iso- and anteiso-branched. Physiological and biochemical characteristics allowed a further phenotypic differentiation of strains JJ-7T and JJ-60T from the most closely related species. Thus, each strain represents a novel species of the genus Paenibacillus, for which the names Paenibacillus auburnensis sp. nov. and Paenibacillus pseudetheri sp. nov. are proposed, with JJ-7T (=CIP 111892T=DSM 111785T=LMG 32088T=CCM 9087T) and JJ-60T (=CIP 111894T=DSM 111787T=LMG 32090T=CCM 9086T) as the type strains, respectively.