Characterization of the emerging zoonotic pathogen Arcobacter thereius by whole genome sequencing and comparative genomics.

Four Arcobacter species have been associated with human disease, and based on current knowledge, these Gram negative bacteria are considered as potential food and waterborne zoonotic pathogens. At present, only the genome of the species Arcobacter butzleri has been analysed, and still little is known about their physiology and genetics. The species Arcobacter thereius has first been isolated from tissue of aborted piglets, duck and pig faeces, and recently from stool of human patients with enteritis. In the present study, the complete genome and analysis of the A. thereius type strain LMG24486T, as well as the comparative genome analysis with 8 other A. thereius strains are presented. Genome analysis revealed metabolic pathways for the utilization of amino acids, which represent the main source of energy, together with the presence of genes encoding for respiration-associated and chemotaxis proteins. Comparative genome analysis with the A. butzleri type strain RM4018 revealed a large correlation, though also unique features. Furthermore, in silico DDH and ANI based analysis of the nine A. thereius strains disclosed clustering into two closely related genotypes. No discriminatory differences in genome content nor phenotypic behaviour were detected, though recently the species Arcobacter porcinus was proposed to encompass part of the formerly identified Arcobacter thereius strains. The report of the presence of virulence associated genes in A. thereius, the presence of antibiotic resistance genes, verified by in vitro susceptibility testing, as well as other pathogenic related relevant features, support the classification of A. thereius as an emerging pathogen.

Whole-Genome Sequence Analysis of Bombella intestini LMG 28161T, a Novel Acetic Acid Bacterium Isolated from the Crop of a Red-Tailed Bumble Bee, Bombus lapidarius.

The whole-genome sequence of Bombella intestini LMG 28161T, an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161T was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161T genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host.

Assessment of the contribution of cocoa-derived strains of Acetobacter ghanensis and Acetobacter senegalensis to the cocoa bean fermentation process through a genomic approach.

Acetobacter ghanensis LMG 23848(T) and Acetobacter senegalensis 108B are acetic acid bacteria that originate from a spontaneous cocoa bean heap fermentation process and that have been characterised as strains with interesting functionalities through metabolic and kinetic studies. As there is currently little genetic information available for these species, whole-genome sequencing of A. ghanensis LMG 23848(T) and A. senegalensis 108B and subsequent data analysis was performed. This approach not only revealed characteristics such as the metabolic potential and genomic architecture, but also allowed to indicate the genetic adaptations related to the cocoa bean fermentation process. Indeed, evidence was found that both species possessed the genetic ability to be involved in citrate assimilation and displayed adaptations in their respiratory chain that might improve their competitiveness during the cocoa bean fermentation process. In contrast, other properties such as the dependence on glycerol or mannitol and lactate as energy sources or a less efficient acid stress response may explain their low competitiveness. The presence of a gene coding for a proton-translocating transhydrogenase in A. ghanensis LMG 23848(T) and the genes involved in two aromatic compound degradation pathways in A. senegalensis 108B indicate that these strains have an extended functionality compared to Acetobacter species isolated from other ecosystems.

Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes.

BACKGROUND: Lactobacillus fermentum 222 and Lactobacillus plantarum 80, isolates from a spontaneous Ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. Lactobacillus fermentum 222 is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. Lactobacillus plantarum 80 is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. In this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process. RESULTS: Through functional annotation and analysis of the high-coverage contigs obtained through 454 pyrosequencing, plantaricin production was predicted for L. plantarum 80. For L. fermentum 222, genes encoding a complete arginine deiminase pathway were attributed. Further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. A comparative genome sequence analysis using publicly available genome sequences of strains of the species L. plantarum and L. fermentum revealed unique features of both strains studied. Indeed, L. fermentum 222 possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas L. plantarum 80 is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose. CONCLUSIONS: In-depth genome sequence analysis of the candidate functional starter culture strains L. fermentum 222 and L. plantarum 80 revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. Further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes.

Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample.

A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures.

Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem.

BACKGROUND: Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. RESULTS: The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. CONCLUSIONS: Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.

Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity.

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.

Alternative antimicrobial compounds to control potential Lactobacillus contamination in bioethanol fermentations.

Antibiotics are commonly used to control microbial contaminants in yeast-based bioethanol fermentation. Given the increase in antibiotic-resistant bacteria, alternative natural antimicrobials were evaluated against the potential contaminant, Lactobacillus. The effects of nisin, ϵ-polylysine, chitosan (CS) and lysozyme were screened against 5 Lactobacillus strains. A standard broth- microdilution method was used in 96-well plates to assess the minimal inhibitory concentration (MIC). L. delbrueckii subsp lactis ATCC479 exhibited maximal MICs with CS, ϵ-polylysine and nisin (1.87, 0.3125 and 0.05 mg/mL, respectively). Nisin reduced most Lactobacillus strains by 6 log CFU/mL after 48 hours with the exception of L. casei. Synergism occurred when ethylenediaminetetraacetic acid (EDTA) was added with nisin. An MIC of 0.4 mg/mL of nisin combined with the EDTA at an MIC of 1 mg/ml markedly suppressed L .casei by 6 log CFU/mL. In conclusion, alternative antimicrobials proved to be a potential candidate for controlling bacterial contamination in the fermentation process. Synergistic effect of nisin with EDTA successfully inhibited the nisin-resistant contaminant, L. casei.