Effects of Chinese medicine herbal residues on antibiotic resistance genes and the bacterial community in chicken manure composting.

The use of livestock manure is an important way for antibiotic resistance genes (ARGs) to enter the environment, and composting is an effective method for removing ARGs from livestock manure. In this study, different volume ratios of Chinese medicinal herbal residues (CMHRs) were added to laboratory-scale chicken manure composting to evaluate their effects, if any, on the behavior of ARGs, mobile genetic elements (MGEs), and the bacterial community. At the end of the composting period, the composition of the microbial community changed. Firmicutes decreased and Bacteroidetes increased. The most striking effect was that the relative abundance of the 21 ARGs and 5 MGEs detected decreased by varying degrees in the different treatments (except for sulI and intI1). The removal rate of the ARGs increased with the increased addition of CMHRs. The correlations between transferase genes (tnpA and tnpA-02) and ARGs were significant (p < 0.05); therefore, transposons play an important role in the horizontal gene transfer of ARGs in chicken manure. The results imply that CMHRs would be an effective bulking agent for the removal of ARGs from chicken manure composting.

Virus-mediated export of chromosomal DNA in plants.

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species.

Uncommon carbapenemase-encoding plasmids in the clinically emergent Acinetobacter pittii.

OBJECTIVES: Two carbapenemase-carrying plasmids, pLS488 (blaOXA-23) and pLS535 (blaOXA-58) from Acinetobacter pittii clinical isolates, were characterized in this study, including their ability to be transferred to Acinetobacter baumannii. METHODS: The clinical isolates were obtained from drainage fluid of a patient with biliary tract cancer and from an exudate of a patient with a hip infection (Portuguese University Hospital, 2012). Isolate characterization included antimicrobial susceptibility tests, carbapenemase production by Blue-Carba, carbapenem-hydrolysing class D ß-lactamase (CHDL) gene search by PCR sequencing, ApaI-PFGE, CHDL genetic location and plasmid size by hybridization and WGS. Plasmid transfer was performed by conjugation or electroporation. RESULTS: pLS488 constitutes the first conjugative plasmid reported to carry a carbapenem resistance gene in A. pittii and is part of a potential new incompatibility group that might also account for the dissemination of OXA-23 in A. baumannii. pLS535 belongs to the Acinetobacter GR7 incompatibility group and presents a new scaffold for OXA-58. This plasmid lacked the machinery for conjugation, but was transferable by electroporation to A. baumannii. Both isolates, which displayed the same PFGE pattern, represent the first report of CHDL-carrying A. pittii in Portuguese hospitals. CONCLUSIONS: Altogether, these results emphasize the importance of A. pittii, or particular A. pittii clones, as a source of resistance genes, facilitating their dissemination among different bacterial species.

Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria.

Mobile genomic islands distribute functional traits between microbes and habitats, yet it remains unclear how their proteins adapt to new environments. Here we used a comparative phylogenomic and proteomic approach to show that the marine bacterium Pseudoalteromonas haloplanktis ANT/505 acquired a genomic island with a functional pathway for pectin catabolism. Bioinformatics and biochemical experiments revealed that this pathway encodes a series of carbohydrate-active enzymes including two multi-modular pectate lyases, PelA and PelB. PelA is a large enzyme with a polysaccharide lyase family 1 (PL1) domain and a carbohydrate esterase family 8 domain, and PelB contains a PL1 domain and two carbohydrate-binding domains of family 13. Comparative phylogenomic analyses indicate that the pathway was most likely acquired from terrestrial microbes, yet we observed multi-modular orthologues only in marine bacteria. Proteomic experiments showed that P. haloplanktis ANT/505 secretes both pectate lyases into the environment in the presence of pectin. These multi-modular enzymes may therefore represent a marine innovation that enhances physical interaction with pectins to reduce loss of substrate and enzymes by diffusion. Our results revealed that marine bacteria can catabolize pectin, and highlight enzyme fusion as a potential adaptation that may facilitate microbial consumption of polymeric substrates in aquatic environments.

Diversification of broad host range plasmids correlates with the presence of antibiotic resistance genes.

The IncP-1ε subgroup is a recently identified phylogenetic clade within IncP-1 plasmids, which plays an important role in the spread of antibiotic resistance and degradation of xenobiotic pollutants. Here, four IncP-1ε plasmids were exogenously captured from a petroleum-contaminated habitat in China and compared phylogenetically and genomically with previously reported IncP-1ε and other IncP-1 plasmids. The IncP-1ε plasmids can be clearly subdivided into two subclades, designated as ε-I and ε-II, based on phylogenetic analysis of backbone proteins TraI and TrfA. This was further supported by comparison of concatenated backbone genes. Moreover, the two subclades differed in the transposon types, phenotypes and insertion locations of the accessory elements. The accessory genes on ε-I plasmids were inserted between parA and traC, and harbored ISPa17 and Tn402-like transposon modules, typically carrying antibiotic resistance genes. In contrast, the accessory elements on ε-II plasmids were typically located between trfA and oriV, and contained IS1071, which was commonly inserted within the Tn501-like transposon, typically harboring a cluster of genes encoding mercury resistance and/or catabolic pathways. Our study is one of the first to compare IncP-1 plasmid genomes from China, expands the available collection of IncP-1ε plasmids and enhances our understanding of their diversity, biogeography and evolutionary history.

Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani.

BACKGROUND: Dickeya solani is an emerging pathogen that causes soft rot and blackleg diseases in several crops including Solanum tuberosum, but little is known about its genomic diversity and evolution. RESULTS: We combined Illumina and PacBio technologies to complete the genome sequence of D. solani strain 3337 that was used as a reference to compare with 19 other genomes (including that of the type strain IPO2222(T)) which were generated by Illumina technology. This population genomic analysis highlighted an unexpected variability among D. solani isolates since it led to the characterization of two distinct sub-groups within the D. solani species. This approach also revealed different types of variations such as scattered SNP/InDel variations as well as replacing and additive horizontal gene transfers (HGT). Infra-species (between the two D. solani sub-groups) and inter-species (between D. solani and D. dianthicola) replacing HGTs were observed. Finally, this work pointed that genetic and functional variation in the motility trait could contribute to aggressiveness variability in D. solani. CONCLUSIONS: This work revealed that D. solani genomic variability may be caused by SNPs/InDels as well as replacing and additive HGT events, including plasmid acquisition; hence the D. solani genomes are more dynamic than that were previously proposed. This work alerts on precautions in molecular diagnosis of this emerging pathogen.

Phylogenetic relationships and the occurrence of interspecific recombination between beet chlorosis virus (BChV) and Beet mild yellowing virus (BMYV).

Samples containing two viruses belonging to the genus Polerovirus, beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV), were collected from French and Polish sugar beet fields. The molecular properties of 24 isolates of BChV and BMYV were investigated, and their genetic diversity was examined in the coat protein (CP)- and P0-encoding genes. For the first time, we have demonstrated that beet polerovirus populations include recombinants between BChV and BMYV containing breakpoints within the CP gene. Moreover, a partial correlation between geographic origin and phylogenetic clustering was observed for BMYV isolates.

Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome.

The mammalian gut ecosystem has considerable influence on host physiology, but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to the gut ecosystem, such as through antibiotic treatment or diet, are at present interpreted at the level of bacterial phylogeny. Less is known about the contributions of the abundant population of phages to this ecological network. Here we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine faecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug, as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phages from treated mice provide aerobically cultured naive microbiota with increased resistance. Systems-wide analyses uncovered post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome becomes broadly enriched for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance, and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress.

Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera.

Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment.

Chromosome pairing affinities in interspecific hybrids reflect phylogenetic distances among lady's slipper orchids (Paphiopedilum).

BACKGROUND AND AIMS: Lady's slipper orchids (Paphiopedilum) are of high value in floriculture, and interspecific hybridization has long been used for breeding improved cultivars; however, information regarding the genome affinities of species and chromosome pairing behaviour of the hybrids remains almost unknown. The present work analyses the meiotic behaviour of interspecific hybrids by genomic in situ hybridization and cytologically evaluates the genomic relationships among parental species. METHODS: Eight interspecific F(1) hybrids of Paphiopedilum species in various subgenera or sections were investigated in this study. The chromosome behaviour in meiosis of these interspecific hybrids was analysed and subjected to genomic in situ hybridization and fluorescent in situ hybridization. KEY RESULTS: Genomic in situ hybridization was demonstrated as an efficient method to differentiate between Paphiopedilum genomes and to visualize the chromosome pairing affinities in interspecific F(1) hybrids, clarifying the phylogenetic distances among these species. Comparatively regular chromosome pairing observed in the hybrids of P. delenatii × P. bellatulum, P. delenatii × P. rothschildianum and P. rothschildianum × P. bellatulum suggested high genomic affinities and close relationships between parents of each hybrid. In contrast, irregular chromosome associations, such as univalents, trivalents and quadrivalents occurred frequently in the hybrids derived from distant parents with divergent karyotypes, such as P. delenatii × P. callosum, P. delenatii × P. glaucophyllum, P. rothschildianum × P. micranthum and P. rothschildianum × P. moquetteanum. The existence of multivalents and autosyndesis demonstrated by genomic in situ hybridization in this study indicates that some micro-rearrangements and other structural alterations may also play a part in differentiating Paphiopedilum species at chromosomal level, demonstrated as different chromosome pairing affinities in interspecific hybrids. CONCLUSIONS: The results indicate that genome homology and the interaction of genetic factors, but not chromosome number nor karyotype similarity, determine the chromosome pairing behaviour in Paphiopedilum hybrids.

In search of the DFNA11 myosin VIIA low- and mid-frequency auditory genetic modifier.

OBJECTIVES: To evaluate the auditory, vestibular, and retinal characteristics of a large American DFNA11 pedigree with autosomal dominant progressive sensorineural hearing loss that first impacts the low- and mid-frequency auditory range. The pedigree (referred to as the HL2 family) segregates a myosin VIIA (MYO7A) mutation in exon 17 at DNA residue G2164C (MYO7A) that seems to be influenced by a genetic modifier that either rescues or exacerbates the MYO7A alteration. DNA analysis to examine single-nucleotide polymorphisms in 2 candidate modifier genes (ATP2B2 and Wolfram syndrome 1 [WFS1]) is summarized in this report. STUDY DESIGN: Family study. RESULTS: The degree of low- and mid-frequency hearing loss in HL2 family members segregating the MYO7A mutation varies from mild to more severe, with approximately the same number of HL2 family members falling at each end of the severity spectrum. The extent of hearing loss in HL2 individuals can vary between family generations. Differences in the degree of hearing loss in MYO7A HL2 family members may be mirrored by vestibular function in at least 2 of these same individuals. The single-nucleotide polymorphisms examined within ATP2B2 and WFS1 did not segregate with the mild versus more severe auditory phenotype. CONCLUSION: The severity of the auditory and vestibular phenotypes in MYO7A HL2 family members may run in parallel, suggesting a common modifier gene within the inner ear. The putative MYO7A genetic modifier is likely to represent a common polymorphism that is not linked tightly to the MYO7A mutation on the MYO7A allele.

Field study results on the probability and risk of a horizontal gene transfer from transgenic herbicide-resistant oilseed rape pollen to gut bacteria of bees.

Bees are specifically subjected to intimate contacts with transgenic plants due to their feeding activities on pollen. In this study, the probability and ecological risk of a gene transfer from pollen to gut bacteria of bees was investigated with larvae of Apis mellifera (honeybee), Bombus terrestris (bumblebee), and Osmia bicornis (red mason bee), all collected at a flowering transgenic oilseed rape field. The plants were genetically engineered with the pat-gene, conferring resistance against glufosinate (syn. phosphinothricin), a glutamine-synthetase inhibitor in plants and microorganisms. Ninety-six bacterial strains were isolated and characterized by 16S rRNA gene sequencing, revealing that Firmicutes represented 58% of the isolates, Actinobacteria 31%, and Proteobacteria 11%, respectively. Of all isolates, 40% were resistant to 1 mM glufosinate, and 11% even to 10 mM. Resistant phenotypes were found in all phylogenetic groups. None of the resistant phenotypes carried the recombinant pat-gene in its genome. The threshold of detecting gene transfer in this field study was relatively insensitive due to the high background of natural glufosinate resistance. However, the broad occurrence of glufosinate-resistant bacteria from different phylogenetic groups suggests that rare events of horizontal gene transfer will not add significantly to natural bacterial glufosinate resistance.

Lessons we can learn from ecological biosafety research.

The last decade has seen an increasing number of biosafety related publications focusing on transgenic organisms. Recent extensive field studies suggest that harmful laboratory effects on non-target organisms rarely occur in the environment. Moreover, biosafety studies typically show no difference in hybridisation between genetically modified plants (GMPs) or non-GMPs and related wild species. Since risk is a product of both exposure and hazard, biosafety research should clearly not only target gene flow exposure but specifically concentrate on expected hazards emerging from successful transgene flow to wild relatives of GMPs. Generally, transgenic plants behave in an ecologically similar manner to non-GMPs if the modified trait confers a neutral advantage under environmental or experimental conditions. However, GMPs perform better than non-GMPs if the new phenotype is challenged by conditions ecologically advantageous for the modified trait. Since biosafety research is a laborious process it will have to concentrate resources on thoughtful, thorough experiments, and target ecologically 'riskier' organisms. So far, we have no evidence that the use of GMPs contradicts sustainable agriculture and nature conservation per se.