Partial replacement of soybean meal with full-fat black soldier fly larvae meal in plant-based nursery diets did not influence fecal Escherichia coli colony forming units or improve fecal consistency when pigs were weaned into non-disinfected pens.

At weaning, one hundred pigs (21 d of age; 6.96 ±â€…0.23 kg BW) were used to determine the effect of partially replacing soybean meal (SBM) in corn- and SBM-based nursery diets on growth performance, fecal scores, Escherichia coli (E. coli) colony forming units (CFU), and cecal mucosal microbial profile when weaned into non-disinfected nursery pens. Pens were randomly assigned to one of four dietary treatments (n = 5): high-complexity (contained highly digestible animal proteins and 10.8% SBM) with and without 3,000 ppm ZnO (HC + and HC-, respectively; representative of commercial diets), low-complexity (corn- and SBM-based; 31.8% SBM; LC), or LC with 30% inclusion of full-fat black soldier fly larvae meal (BSFLM) to partially replace SBM (LCFL; 8.0% SBM). Diets were fed for 14 d (phase I), followed by 4 wk of a common corn-SBM diet (phase II). Fecal E. coli CFU and cecal mucosal microbial 16s rRNA community profiles were assessed 7 d after weaning. During phase I, pigs fed LC and LCFL had lower average daily gains (P < 0.05) than pigs fed HC + and HC-, which were not different. Average daily feed intake was not different for pigs fed LC and LCFL, but lower than for pigs fed HC- (P < 0.001); pigs fed HC + had greater feed intake in phase I vs. all other treatment groups (P < 0.001). Upon nursery exit, only pigs fed LCFL had lower BW than pigs fed HC- (P < 0.05), with intermediate values observed for HC + and LC. Day 3 fecal scores were greater for pigs fed LCFL vs. HC + (P < 0.05) and day 7 E. coli CFU were greater for all treatment groups vs. HC + (P < 0.001). Pigs fed HC- (P < 0.01), LC (P < 0.05), and LCFL (P < 0.05) had lower alpha diversity for cecal mucosal microbiota compared to HC+. At the genus level, pigs fed LC had lower Lactobacillus relative abundance vs. pigs fed HC + (P < 0.01). Therefore, BSFLM can partially replace SBM without sacrificing growth performance vs. nursery pigs fed corn- and SBM-based diets, but both groups had reduced phase I growth performance vs. pigs fed highly digestible diets containing animal proteins when weaned into non-disinfected pens. The BSFLM did not influence fecal E. coli CFU or improve fecal consistency after weaning and therefore, is less effective at minimizing digestive upsets vs. HC + diets.

Incorporating the plasmidome into antibiotic resistance surveillance in animal agriculture.

Mobile genetic elements (MGE) carrying resistance genes represent a unique challenge to risk assessment and surveillance of antimicrobial resistance (AMR). Yet determining the mobility of resistance genes within animal microbiomes is essential to evaluating the potential dissemination from livestock to potential human pathogens, as well as evaluating co-selection mechanisms that may impact persistence of resistance genes with changing antibiotic use patterns. Current surveillance efforts utilize phenotypic testing and sequencing of individual isolates for tracking of AMR in livestock. In this work, we investigated the utility of using long-read sequencing of the plasmids from mixed Enterobacterales enrichments of swine fecal samples as a surveillance strategy for AMR plasmids. Enrichments were performed in either MacConkey broth without selection or with selection by addition of tetracycline or ceftriaxone, and plasmids were extracted and sequenced in order to evaluate the diversity of plasmids enriched by each method. Intact resistance plasmids were successfully assembled, as well as complex resistance transposons carrying multiple repeated elements that would interfere with assembly by short read sequencing technologies. Comparison of the assembled plasmids with representatives from public databases confirmed the quality of the assemblies and also revealed the occurrence of IncI2 plasmids carrying blaCMY-2 in Ontario swine samples, which have not been found in previous studies.

PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies.

Phylogeny and organization of recombinase in trio (RIT) elements.

Recombinase in trio (RIT) elements are composed of three adjacent tyrosine based site-specific recombinases that commonly occur in bacterial genomes. In this study, we examine RIT elements found in the genomes of strains from 63 different genera across 7 phyla of Eubacteria and examine the specific organization of these elements, their phylogenetic and environmental distribution, and their potential for mobility. We have found that each recombinase in this RIT arrangement is associated with a distinct sub-family of the tyrosine recombinases, and that the order and orientation of these sub-families is consistently maintained. We have determined that the distribution of these elements suggests that they are an ancient feature of bacterial genomes, but identical copies found within individual strains indicates that they are capable of intragenomic mobility. The occurrence of identical elements on both the main chromosome and one or more plasmids within individual strains, coupled with the finding that in some cases related genera are carrying highly similar RIT elements indicates that horizontal transfer has in some cases proceeded through a plasmid intermediate.

The limitations of draft assemblies for understanding prokaryotic adaptation and evolution.

The de novo assembly of next generation sequencing data is a daunting task made more difficult by the presence of genomic repeats or transposable elements, resulting in an increasing number of genomes designated as completed draft assemblies. We created and assembled idealized sequence data sets for Cupriavidus metallidurans CH34, Caulobacter sp. K31, Gramella forsetii KT0803, Rhodobacter sphaeroides 2.4.1 and Bordetella bronchiseptica RB50. In addition to confirming the role of transposable elements in interrupting the assemblies, an association was found between the most fragmented regions and known or predicted genomic islands in these strains. Assembly quality was more strongly related to putative genomic island content than to any other factor examined. We believe this association indicates that draft assemblies are limiting our ability to understand the genomic context of important bacterial adaptations and that the increased effort required for finishing genomes can provide a wealth of information for future studies.