Honey bees harbor a diverse gut virome engaging in nested strain-level interactions with the microbiota.

The honey bee gut microbiota influences bee health and has become an important model to study the ecology and evolution of microbiota-host interactions. Yet, little is known about the phage community associated with the bee gut, despite its potential to modulate bacterial diversity or to govern important symbiotic functions. Here we analyzed two metagenomes derived from virus-like particles, analyzed the prevalence of the identified phages across 73 bacterial metagenomes from individual bees, and tested the host range of isolated phages. Our results show that the honey bee gut virome is composed of at least 118 distinct clusters corresponding to both temperate and lytic phages and representing novel genera with a large repertoire of unknown gene functions. We find that the phage community is prevalent in honey bees across space and time and targets the core members of the bee gut microbiota. The large number and high genetic diversity of the viral clusters seems to mirror the high extent of strain-level diversity in the bee gut microbiota. We isolated eight lytic phages that target the core microbiota member Bifidobacterium asteroides, but that exhibited different host ranges at the strain level, resulting in a nested interaction network of coexisting phages and bacterial strains. Collectively, our results show that the honey bee gut virome consists of a complex and diverse phage community that likely plays an important role in regulating strain-level diversity in the bee gut and that holds promise as an experimental model to study bacteria-phage dynamics in natural microbial communities.

Bloom of resident antibiotic-resistant bacteria in soil following manure fertilization.

The increasing prevalence of antibiotic-resistant bacteria is a global threat to public health. Agricultural use of antibiotics is believed to contribute to the spread of antibiotic resistance, but the mechanisms by which many agricultural practices influence resistance remain obscure. Although manure from dairy farms is a common soil amendment in crop production, its impact on the soil microbiome and resistome is not known. To gain insight into this impact, we cultured bacteria from soil before and at 10 time points after application of manure from cows that had not received antibiotic treatment. Soil treated with manure contained a higher abundance of ß-lactam-resistant bacteria than soil treated with inorganic fertilizer. Functional metagenomics identified ß-lactam-resistance genes in treated and untreated soil, and indicated that the higher frequency of resistant bacteria in manure-amended soil was attributable to enrichment of resident soil bacteria that harbor ß-lactamases. Quantitative PCR indicated that manure treatment enriched the blaCEP-04 gene, which is highly similar (96%) to a gene found previously in a Pseudomonas sp. Analysis of 16S rRNA genes indicated that the abundance of Pseudomonas spp. increased in manure-amended soil. Populations of other soil bacteria that commonly harbor ß-lactamases, including Janthinobacterium sp. and Psychrobacter pulmonis, also increased in response to manure treatment. These results indicate that manure amendment induced a bloom of certain antibiotic-resistant bacteria in soil that was independent of antibiotic exposure of the cows from which the manure was derived. Our data illustrate the unintended consequences that can result from agricultural practices, and demonstrate the need for empirical analysis of the agroecosystem.

Transcriptional responses of Italian ryegrass during interaction with Xanthomonas translucens pv. graminis reveal novel candidate genes for bacterial wilt resistance.

Xanthomonas translucens pv. graminis (Xtg) causes bacterial wilt, a severe disease of forage grasses such as Italian ryegrass (Lolium multiflorum Lam.). In order to gain a more detailed understanding of the genetic control of resistance mechanisms and to provide prerequisites for marker assisted selection, the partial transcriptomes of two Italian ryegrass genotypes, one resistant and one susceptible to bacterial wilt were compared at four time points after Xtg infection. A cDNA microarray developed from a perennial ryegrass (Lolium perenne) expressed sequence tag set consisting of 9,990 unique genes was used for transcriptome analysis in Italian ryegrass. An average of 4,487 (45%) of the perennial ryegrass sequences spotted on the cDNA microarray were detected by cross-hybridisation to Italian ryegrass. Transcriptome analyses of the resistant versus the susceptible genotype revealed substantial gene expression differences (>1,200) indicating that great gene expression differences between different Italian ryegrass genotypes exist which potentially contribute to the observed phenotypic divergence in Xtg resistance between the two genotypes. In the resistant genotype, several genes differentially expressed after Xtg inoculation were identified which revealed similarities to transcriptional changes triggered by pathogen-associated molecular patterns in other plant-pathogen interactions. These genes represent candidate genes of particular interest for the development of tools for marker assisted resistance breeding.

Corrigendum: Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

[This corrects the article DOI: 10.3389/fmicb.2017.02675.].

Monitoring of genetically modified Escherichia coli in laboratory wastewater.

Containment of genetically modified (GM) microorganisms such as Escherichia coli is a legal requirement to protect the environment from an unintended release and to avoid horizontal gene transfer (HGT) of recombinant DNA to native bacteria. In this study, we sampled the laboratory wastewater (LWW) at a large Swiss university from three sources over 2 years and cultured ampicillin-resistant, presumptive GM E. coli. From a total of 285 samples, 127 contained presumptive GM E. coli (45%) at a mean concentration of 2.8 × 102 CFU/ml. Plasmid DNA of 11 unique clones was partially or entirely sequenced. All consisted of cloning vectors harboring research-specific inserts. To estimate the chance of HGT between GM E. coli and native bacteria in LWW, we identified taxa representative for the bacterial community in LWW using 16S rRNA amplicon sequencing and measured conjugation frequencies of E. coli with five LWW isolates. At optimal conjugation conditions, frequencies were between 3.4 × 10-3 and 2.4 × 10-5. Given the absence of transferable broad-host range plasmids and suboptimal conjugation conditions in the LWW system, we conclude that the chance of HGT is relatively low. Still, this study shows that the implementation of robust containment measures is key to avoid the escape of GM microorganisms.

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents.

KP4 to control Ustilago tritici in wheat: Enhanced greenhouse resistance to loose smut and changes in transcript abundance of pathogen related genes in infected KP4 plants.

Ustilago tritici causes loose smut, which is a seed-borne fungal disease of wheat, and responsible for yield losses up to 40%. Loose smut is a threat to seed production in developing countries where small scale farmers use their own harvest as seed material. The killer protein 4 (KP4) is a virally encoded toxin from Ustilago maydis and inhibits growth of susceptible races of fungi from the Ustilaginales. Enhanced resistance in KP4 wheat to stinking smut, which is caused by Tilletia caries, had been reported earlier. We show that KP4 in genetically engineered wheat increased resistance to loose smut up to 60% compared to the non-KP4 control under greenhouse conditions. This enhanced resistance is dose and race dependent. The overexpression of the transgene kp4 and its effect on fungal growth have indirect effects on the expression of endogenous pathogen defense genes.

Diverse antibiotic resistance genes in dairy cow manure.

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection.

Xanthomonas translucens pv. graminis (Xtg) is a gammaproteobacterium that causes bacterial wilt on a wide range of forage grasses. To gain insight into the host-pathogen interaction and to identify the virulence factors of Xtg, we compared a draft genome sequence of one isolate (Xtg29) with other Xanthomonas spp. with sequenced genomes. The type III secretion system (T3SS) encoding a protein transport system for type III effector (T3E) proteins represents one of the most important virulence factors of Xanthomonas spp. In contrast with other Xanthomonas spp. assigned to clade 1 on the basis of phylogenetic analyses, we identified an hrp (hypersensitive response and pathogenicity) gene cluster encoding T3SS components and a representative set of 35 genes encoding putative T3Es in the genome of Xtg29. The T3SS was shown to be divergent from the hrp gene clusters of other sequenced Xanthomonas spp. Xtg mutants deficient in T3SS regulating and structural genes were constructed to clarify the role of the T3SS in forage grass colonization. Italian ryegrass infection with these mutants led to significantly reduced symptoms (P < 0.05) relative to plants infected with the wild-type strain. This showed that the T3SS is required for symptom evocation. In planta multiplication of the T3SS mutants was not impaired significantly relative to the wild-type, indicating that the T3SS is not required for survival until 14 days post-infection. This study represents the first major step to understanding the bacterial colonization strategies deployed by Xtg and may assist in the identification of resistance (R) genes in forage grasses.