DNA metabarcoding reveals the impact of Cu2+ on soil cercozoan diversity.

Although copper (Cu2+) is a micronutrient, the metal may be toxic if present in high concentrations in soil ecosystems and subsequently affect various organisms, ranging from microorganisms to earthworms. We performed a microcosm study with an array of Cu2+ concentrations, with a specific focus on Cercozoa, an important protozoan group in most soil food webs. Research on Cercozoa is still scarce in terms of both diversity and ecology; hence, to explore this group in more depth, we used high-throughput sequencing to detect Cu2+ induced community changes. Increased levels of Cu2+ caused a shift in the cercozoan community, and we observed decreased cercozoan relative abundance across the majority of orders, families and genera. Due to their key role in soil food webs, especially as bacterial predators and providers of nutrients to plants, the reduction of cercozoan abundance and diversity may seriously affect soil functionality. Our results indicate that the increase of Cu2+ concentrations in the soil could potentially have this effect and the consequences need exploration.

LotuS2: an ultrafast and highly accurate tool for amplicon sequencing analysis.

BACKGROUND: Amplicon sequencing is an established and cost-efficient method for profiling microbiomes. However, many available tools to process this data require both bioinformatics skills and high computational power to process big datasets. Furthermore, there are only few tools that allow for long read amplicon data analysis. To bridge this gap, we developed the LotuS2 (less OTU scripts 2) pipeline, enabling user-friendly, resource friendly, and versatile analysis of raw amplicon sequences. RESULTS: In LotuS2, six different sequence clustering algorithms as well as extensive pre- and post-processing options allow for flexible data analysis by both experts, where parameters can be fully adjusted, and novices, where defaults are provided for different scenarios. We benchmarked three independent gut and soil datasets, where LotuS2 was on average 29 times faster compared to other pipelines, yet could better reproduce the alpha- and beta-diversity of technical replicate samples. Further benchmarking a mock community with known taxon composition showed that, compared to the other pipelines, LotuS2 recovered a higher fraction of correctly identified taxa and a higher fraction of reads assigned to true taxa (48% and 57% at species; 83% and 98% at genus level, respectively). At ASV/OTU level, precision and F-score were highest for LotuS2, as was the fraction of correctly reported 16S sequences. CONCLUSION: LotuS2 is a lightweight and user-friendly pipeline that is fast, precise, and streamlined, using extensive pre- and post-ASV/OTU clustering steps to further increase data quality. High data usage rates and reliability enable high-throughput microbiome analysis in minutes. AVAILABILITY: LotuS2 is available from GitHub, conda, or via a Galaxy web interface, documented at http://lotus2.earlham.ac.uk/ . Video Abstract.

Comparative genomics of Rothia species reveals diversity in novel biosynthetic gene clusters and ecological adaptation to different eukaryotic hosts and host niches.

Rothia species are understudied members of the phylum Actinobacteria and prevalent colonizers of the human and animal upper respiratory tract and oral cavity. The oral cavity, including the palatine tonsils, is colonized by a complex microbial community, which compete for resources, actively suppress competitors and influence host physiology. We analysed genomic data from 43 new porcine Rothia isolates, together with 112 publicly available draft genome sequences of Rothia isolates from humans, animals and the environment. In all Rothia genomes, we identified biosynthetic gene clusters predicted to produce antibiotic non-ribosomal peptides, iron scavenging siderophores and other secondary metabolites that modulate microbe-microbe and potentially microbe-host interactions. In vitro overlay inhibition assays corroborated the hypothesis that specific strains produce natural antibiotics. Rothia genomes encode a large number of carbohydrate-active enzymes (CAZy), with varying CAZy activities among the species found in different hosts, host niches and environments. These findings reveal competition mechanisms and metabolic specializations linked to ecological adaptation of Rothia species in different hosts.

Impact of the Gram-Negative-Selective Inhibitor MAC13243 on In Vitro Simulated Gut Microbiota.

New Gram-negative-selective antimicrobials are desirable to avoid perturbations in the gut microbiota leading to antibiotic-induced dysbiosis. We investigated the impact of a prototype drug (MAC13243) interfering with the Gram-negative outer membrane protein LolA on the faecal microbiota. Faecal suspensions from two healthy human donors were exposed to MAC13243 (16, 32, or 64 mg/L) using an in vitro gut model (CoMiniGut). Samples collected 0, 4, and 8 h after exposure were subjected to viable cell counts, 16S rRNA gene quantification and V3-V4 sequencing using the Illumina MiSeq platform. MAC13243 exhibited concentration-dependent killing of coliforms in both donors after 8 h. Concentrations of ≤32 mg/L reduced the growth of aerobic bacteria without influencing the microbiota composition and diversity. An expansion of Firmicutes at the expense of Bacteroidetes and Actinobacteria was observed in the faecal microbiota from one donor following exposure to 64 mg/L of MAC13242. At all concentrations tested, MAC13243 did not lead to the proliferation of Escherichia coli nor a reduced abundance of beneficial bacteria, which are typical changes observed in antibiotic-induced dysbiosis. These results support our hypothesis that a drug interfering with a specific target in Gram-negative bacteria has a low impact on the commensal gut microbiota and, therefore, a low risk of inducing dysbiosis.

Classification of In Vitro Phage-Host Population Growth Dynamics.

The therapeutic use of bacteriophages (phage therapy) represents a promising alternative to antibiotics to control bacterial pathogens. However, the understanding of the phage-bacterium interactions and population dynamics seems essential for successful phage therapy implementation. Here, we investigated the effect of three factors: phage species (18 lytic E. coli-infecting phages); bacterial strain (10 APEC strains); and multiplicity of infection (MOI) (MOI 10, 1, and 0.1) on the bacterial growth dynamics. All factors had a significant effect, but the phage appeared to be the most important. The results showed seven distinct growth patterns. The first pattern corresponded to the normal bacterial growth pattern in the absence of a phage. The second pattern was complete bacterial killing. The remaining patterns were in-between, characterised by delayed growth and/or variable killing of the bacterial cells. In conclusion, this study demonstrates that the phage-host dynamics is an important factor in the capacity of a phage to eliminate bacteria. The classified patterns show that this is an essential factor to consider when developing a phage therapy. This methodology can be used to rapidly screen for novel phage candidates for phage therapy. Accordingly, the most promising candidates were phages found in Group 2, characterised by growth dynamics with high bacterial killing.

Clonal spread of multi-resistant Gallibacterium anatis isolates among Iranian broilers and layers.

Gallibacterium anatis is a common cause of reproductive tract infection in chickens, which leads to reduced egg production and increased mortality. This study was undertaken to investigate prevalence of G. anatis in 12 poultry flocks originating from Iranian provinces with leading chicken production and to determine genetic diversity, antimicrobial resistance, and the presence of major antigens of the isolates investigated. Out of the 120 chicken tracheal samples collected and tested, 84 (70%) were positive for G. anatis. Genotyping by Pulse Field Gel Electrophoresis and genome sequencing revealed a total of 24 pulsotypes for 71 strains (at a 87% similarity level) and seven genome clusters comprising 21 strains (97% similarity level), respectively. The combination of the two typing methods confirmed the presence of several genotypes originating from a common ancestor affecting poultry yet also suggested that identical clones were shared among chickens within farms and between different farms. The latter finding is to our knowledge the first example of clonal presence of G. anatis in epidemiologically unrelated farms. The 21 sequenced strains were characterized against a panel of commonly used antibiotics and showed lowered sensitivity to tetracycline (76.2%) and enrofloxacin (90.5%). The widespread presence of multiresistant G. anatis isolates calls for non-antibiotic prophylactics. Three major immunogen genes, gtxA, Gab_1309 and Gab_2312 were detected in the isolates indicating these antigens likely represent effective vaccine targets. A conserved sequence of the gtxA gene across a range of epidemiologically independent strains suggests the use of GtxA for future vaccine development purposes.

Complete genome of a methicillin-resistant Staphylococcus vitulinus from Danish ground beef meat carrying a mecA2 resistance gene and a novel ccr allotype.

OBJECTIVES: To report the complete genome sequence of a methicillin-resistant Staphylococcus vitulinus from ground beef to allow comparison with other available S. vitulinus genomes and to investigate its SCCmec element. METHODS: Meat samples from grocery stores in Denmark were examined for the presence of staphylococcal species by plating on selective plates. One colony isolated from beef was identified as S. vitulinus by MALDI-TOF and genome sequenced using a combination of Illumina and Oxford Nanopore technologies. Phylogenetic and in silico resistome analyses were performed for all available S. vitulinus genomes. RESULTS: The closed genome of S. vitulinus Tienloo1 isolate had a chromosome size of 2,628,028 bp and contained a single novel 2,380 bp plasmid based on a hybrid assembly. It carried mecA as the only resistance marker. The isolate was found not to carry any immune evasion cluster genes, which have been putatively associated to human origin. Comparison with all publicly available S. vitulinus draft genomes showed a diverse population and revealed that only the Danish beef isolate contained a mec gene in addition to a ccr gene complex. Additionally, the single ccrC gene within the isolate was novel and distant from the mecA2 gene. CONCLUSION: This isolate, Tienloo1, from a ground beef meat sample represents the first complete genome of S. vitulinus found to carry a mecA2 gene and a novel ccr allotype in its SCCmec element that is distinct from all publicly available draft S. vitulinus genomes.