Potential reservoirs of antimicrobial resistance in livestock waste and treated wastewater that can be disseminated to agricultural land.

Livestock manure, dairy lagoon effluent, and treated wastewater are known reservoirs of antibiotic resistance genes (ARGs), antibiotic-resistant bacteria (ARB), and virulence factor genes (VFGs), and their application to agricultural farmland could be a serious public health threat. However, their dissemination to agricultural lands and impact on important geochemical pathways such as the nitrogen (N) cycle have not been jointly explored. In this study, shotgun metagenomic sequencing and analyses were performed to examine the diversity and composition of microbial communities, ARGs, VFGs, and N cycling genes in different livestock manure/lagoon and treated wastewater collected from concentrated animal feeding operations (CAFOs) and a municipal wastewater treatment plant along the west coast of the United States. Multivariate analysis showed that diversity indices of bacterial taxa from the different microbiomes were not significantly different based on InvSimpson (P = 0.05), but differences in ARG mechanisms were observed between swine manure and other microbiome sources. Comparative resistome profiling showed that ARGs in microbiome samples belonged to four core resistance classes: aminoglycosides (40-55 %), tetracyclines (30-45 %), beta-lactam-resistance (20-35 %), macrolides (18-30 %), and >50 % of the VFGs that the 24 microbiomes harbored were phyletically affiliated with two bacteria, Bacteroidetes fragilis and Enterobacter aerogenes. Network analysis based on Spearman correlation showed co-occurrence patterns between several genes such as transporter-gene and regulator, efflux pump and involved-in-polymyxin- resistance, aminoglycoside, beta-lactam, and macrolide with VFGs and bacterial taxa such as Firmicutes, Candidatus Themoplasmatota, Actinobacteria, and Bacteroidetes. Metabolic reconstruction of metagenome-assembled genome (MAGs) analysis showed that the most prevalent drug resistance mechanisms were associated with carbapenem resistance, multidrug resistance (MDR), and efflux pump. Bacteroidales was the main taxa involved in dissimilatory nitrate reduction (DNRA) in dairy lagoon effluent. This study demonstrates that the dissemination of waste from these sources can increase the spread of ARGs, ARB, and VFGs into agricultural lands, negatively impacting both soil and human health.

Microbial community structures in high rate algae ponds for bioconversion of agricultural wastes from livestock industry for feed production.

Dynamics of seasonal microbial community compositions in algae cultivation ponds are complex. However, there is very limited knowledge on bacterial communities that may play significant roles with algae in the bioconversion of manure nutrients to animal feed. In this study, water samples were collected during winter, spring, summer, and fall from the dairy lagoon effluent (DLE), high rate algae ponds (HRAP) that were fed with diluted DLE, and municipal waste water treatment plant (WWTP) effluent which was included as a comparison system for the analysis of total bacteria, Cyanobacteria, and microalgae communities using MiSeq Illumina sequencing targeting the 16S V4 rDNA region. The main objective was to examine dynamics in microbial community composition in the HRAP used for the production of algal biomass. DNA was extracted from the different sample types using three commercially available DNA extraction kits; MoBio Power water extraction kit, Zymo fungi/bacterial extraction kit, and MP Biomedicals FastDNA SPIN Kit. Permutational analysis of variance (PERMANOVA) using distance matrices on each variable showed significant differences (P=0.001) in beta-diversity based on sample source. Environmental variables such as hydraulic retention time (HRT; P<0.031), total N (P<0.002), total inorganic N (P<0.002), total P (P<0.002), alkalinity (P<0.002), pH (P<0.022), total suspended solid (TSS; P<0.003), and volatile suspended solids (VSS; P<0.002) significantly affected microbial communities in DLE, HRAP, and WWTP. Of the operational taxonomic units (OTUs) identified to phyla level, the dominant classes of bacteria identified were: Cyanobacteria, Alpha-, Beta-, Gamma-, Epsilon-, and Delta-proteobacteria, Bacteroidetes, Firmicutes, and Planctomycetes. Our data suggest that microbial communities were significantly affected in HRAP by different environmental variables, and care must be taken in extraction procedures when evaluating specific groups of microbial communities for specific functions.

DNA sequence and genetic characterization of plasmid pFQ11 from Frankia alni strain CpI1.

An 8551-bp plasmid, pFQ11, from Frankia alni strain CpI1 was sequenced. Its sequence was found to be very similar to that presented for pFQ31 from strain ArI3. Six potential protein-encoding open reading frames (ORFs) were identified, and transcriptional activity was shown within four of those regions of the plasmid by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An earlier study reported that ORF E(F) of pFQ31, which is nearly identical to the 3' 45% of ORF1 of pFQ11, is significantly similar to RepF. We found no such similarity. ORF2 and ORF3 predict products that are similar to a repressor protein and a partition protein, respectively. We found inverted repeats within and covering the start codon of ORF3; palindromic sequences and direct repeats between ORF3 and ORF4; and 3' from ORF3, an AT-rich sequence that extensively overlaps the promoter region of a uvrB homolog in strain ArI3.