Pectins are plant cell-wall polysaccharides which can be utilized by commensal bacteria in the gut, exhibiting beneficial properties for the host. Knowledge of the impact of pectins on intestinal bacterial communities is insufficient and limited to a few types of pectins. This study characterized the relationship between the structural properties of pectins and their potential to modulate composition and activity of the gut microbiota in a beneficial way. For this purpose we performed in vitro fermentations of nine structurally diverse pectins from citrus fruits and sugar beet, and a pectic derivative, rhamnogalacturonan I (RGI), using a TIM-2 colon model. The composition of microbiota during TIM-2 fermentations was assessed by 16S rRNA gene amplicon sequencing. Both general and pectin-specific changes were observed in relative abundances of numerous bacterial taxa in a time-dependent way. Bacterial populations associated with human health, such as Faecalibacterium prausnitzii, Coprococcus, Ruminococcus, Dorea, Blautia, Oscillospira, Sutterella, Bifidobacterium, Christensenellaceae, Prevotella copri, and Bacteroides spp. were either increased or decreased depending on the substrate, suggesting that these bacteria can be controlled using structurally different pectins. The main structural features linked to the pectin-mediated shifts in microbiota included degree of esterification, composition of neutral sugars, distribution of homogalacturonan and rhamnogalacturonan fractions, degree of branching, and the presence of amide groups. Cumulative production of the total short chain fatty acids and propionate was largest in fermentations of the high methoxyl pectins. Thus, this study indicates that microbial communities in the gut can be specifically modulated by pectins and identifies the features in pectin molecules linked to microbial alterations. This knowledge can be used to define preferred dietary pectins, targeting beneficial bacteria, and favoring more balanced microbiota communities in the gut.

Background: Metagenomic sequencing is a well-established tool in the modern biosciences. While it promises unparalleled insights into the genetic content of the biological samples studied, conclusions drawn are at risk from biases inherent to the DNA sequencing methods, including inaccurate abundance estimates as a function of genomic guanine-cytosine (GC) contents. Results: We explored such GC biases across many commonly used platforms in experiments sequencing multiple genomes (with mean GC contents ranging from 28.9% to 62.4%) and metagenomes. GC bias profiles varied among different library preparation protocols and sequencing platforms. We found that our workflows using MiSeq and NextSeq were hindered by major GC biases, with problems becoming increasingly severe outside the 45–65% GC range, leading to a falsely low coverage in GC-rich and especially GC-poor sequences, where genomic windows with 30% GC content had >10-fold less coverage than windows close to 50% GC content. We also showed that GC content correlates tightly with coverage biases. The PacBio and HiSeq platforms also evidenced similar profiles of GC biases to each other, which were distinct from those seen in the MiSeq and NextSeq workflows. The Oxford Nanopore workflow was not afflicted by GC bias. Conclusions: These findings indicate potential sources of difficulty, arising from GC biases, in genome sequencing that could be pre-emptively addressed with methodological optimizations provided that the GC biases inherent to the relevant workflow are understood. Furthermore, it is recommended that a more critical approach be taken in quantitative abundance estimates in metagenomic studies. In the future, metagenomic studies should take steps to account for the effects of GC bias before drawing conclusions, or they should use a demonstrably unbiased workflow.

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens isStaphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistantS. aureus(MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range amongS. aureusbacteria. Morphologically, the phage belongs to theMyoviridaefamily and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range ofS. aureusisolates, including representatives of the major international MRSA clones and coagulase-negativeStaphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producingS. aureusisolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused byS. aureusbiofilms.

The human gut is densely populated with archaea, eukaryotes, bacteria, and their viruses, such as bacteriophages. Advances in high-throughput sequencing (HTS) as well as bioinformatics have opened new opportunities for characterizing the viral communities harbored in our gut. However, limited attention has been given to the efficiency of protocols dealing with extraction of phages from fecal communities prior to HTS and their impact on the metagenomic dataset. We describe two optimized methods for extraction of phages from fecal samples based on tangential-flow filtration (TFF) and polyethylene glycol precipitation (PEG) approaches using an adapted method from a published protocol as control (literature-adapted protocol (LIT)). To quantify phage recovery, samples were spiked with low numbers of c2, ϕ29, and T4 phages (representatives of the Siphoviridae, Podoviridae, and Myoviridae families, respectively) and their concentration (plaque-forming units) followed at every step during the extraction procedure. Compared with LIT, TFF and PEG had higher recovery of all spiked phages, yielding up to 16 times more phage particles (PPs) and up to 68 times more phage DNA per volume, increasing thus the chances of extracting low abundant phages. TFF- and PEG-derived metaviromes showed 10 % increase in relative abundance of Caudovirales and unclassified phages infecting gut-associated bacteria (>92 % for TFF and PEG, 82.4 % for LIT). Our methods obtained lower relative abundance of the Myoviridae family (32.5 %, LIT 22.6 %), which was achieved with the enhanced conditions of our procedures (e.g., reduced filter clogging). A high degree of phage diversity in samples extracted using TFF and PEG was documented by transmission electron microscopy. Two procedures (TFF and PEG) for extraction of bacteriophages from fecal samples were optimized using a set of spiked bacteriophages as process control. These protocols are highly efficient tools for extraction and purification of PPs prior to HTS in phage-metavirome studies. Our methods can be easily modified, being thus applicable and adjustable for in principle any solid environmental material in dissolution.

Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G⁺), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2′-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2′-deoxy-7-amido-7-deazaguanine (dADG), 2′-deoxy-7-cyano-7-deazaguanine (dPreQ₀) and 2′-deoxy-7- aminomethyl-7-deazaguanine (dPreQ₁). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2′-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.

Summary: P seudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities – biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P . aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P . aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P . aeruginosa PAO 1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension ( MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P . aeruginosa infections, which are predominantly biofilm centred.