Viral Communities Contribute More to the Lysis of Antibiotic-Resistant Bacteria than the Transduction of Antibiotic Resistance Genes in Anaerobic Digestion Revealed by Metagenomics.

Ecological role of the viral community on the fate of antibiotic resistance genes (ARGs) (reduction vs proliferation) remains unclear in anaerobic digestion (AD). Metagenomics revealed a dominance of Siphoviridae and Podoviridae among 13,895 identified viral operational taxonomic units (vOTUs) within AD, and only 21 of the vOTUs carried ARGs, which only accounted for 0.57 ± 0.43% of AD antibiotic resistome. Conversely, ARGs locating on plasmids and integrative and conjugative elements accounted for above 61.0%, indicating a substantial potential for conjugation in driving horizontal gene transfer of ARGs within AD. Virus-host prediction based on CRISPR spacer, tRNA, and homology matches indicated that most viruses (80.2%) could not infect across genera. Among 480 high-quality metagenome assembly genomes, 95 carried ARGs and were considered as putative antibiotic-resistant bacteria (pARB). Furthermore, lytic phages of 66 pARBs were identified and devoid of ARGs, and virus/host abundance ratios with an average value of 71.7 indicated extensive viral activity and lysis. The infectivity of lytic phage was also elucidated through laboratory experiments concerning changes of the phage-to-host ratio, pH, and temperature. Although metagenomic evidence for dissemination of ARGs by phage transduction was found, the higher proportion of lytic phages infecting pARBs suggested that the viral community played a greater role in reducing ARB numbers than spreading ARGs in AD.

Enrichment of Anaerobic Microbial Communities from Midgut and Hindgut of Sun Beetle Larvae (Pachnoda marginata) on Wheat Straw: Effect of Inoculum Preparation.

The Pachnoda marginata larva have complex gut microbiota capable of the effective conversion of lignocellulosic biomass. Biotechnological utilization of these microorganisms in an engineered system can be achieved by establishing enrichment cultures using a lignocellulosic substrate. We established enrichment cultures from contents of the midgut and hindgut of the beetle larva using wheat straw in an alkaline medium at mesophilic conditions. Two different inoculation preparations were used: procedure 1 (P1) was performed in a sterile bench under oxic conditions using 0.4% inoculum and small gauge needles. Procedure 2 (P2) was carried out under anoxic conditions using more inoculum (4%) and bigger gauge needles. Higher methane production was achieved with P2, while the highest acetic acid concentrations were observed with P1. In the enrichment cultures, the most abundant bacterial families were Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae, and Marinilabiliaceae. Further, the most abundant methanogenic genera were Methanobrevibacter, Methanoculleus, and Methanosarcina. Our observations suggest that in samples processed with P1, the volatile fatty acids were not completely converted to methane. This is supported by the finding that enrichment cultures obtained with P2 included acetoclastic methanogens, which might have prevented the accumulation of acetic acid. We conclude that differences in the inoculum preparation may have a major influence on the outcome of enrichment cultures from the P. marginata larvae gut.

Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum).

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.