Background: Bacteriophages (phages) are the most numerous biological entities on Earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil will shed light not only on the yet largely unknown phage diversity, but may also result in novel insights towards their functioning in the global biogeochemical nutrient cycle and their significance in earthbound ecosystems. Unfortunately, information about soil viromes is rather scarce compared to aquatic environments, due to the heterogeneous soil matrix, which rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is, therefore, a fundamental prerequisite for replicable results and comparative virome studies. Results: We here report the optimization of protocols for the extraction of phage DNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with Listeria phage A511 (Myovirus), Staphylococcus phage 2638AΔLCR (Siphovirus) and Escherichia phage T7 (Podovirus) (each 10 6 PFU/g soil). The efficacy of phage (i) elution, (ii) filtration, (iii) concentration and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for shotgun sequencing devoid of amplification prior library preparation, and low 16S rRNA gene contamination levels (≤ 0.2‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65%. In addition, 379 novel putative complete soil phage genomes (≤ 235 kb) were obtained from over 13,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity. Conclusion: We have shown a considerably enhanced extraction of the soil phage community by protocol optimization that has proven robust in both culture-dependent as well as through viromic analyses. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment.

11Background 12Bacteriophages are the most numerous biological entities on earth and play a crucial role in 13 shaping microbial communities. Investigating the bacteriophage community from soil samples 14 will shed light not only on the yet largely unknown phage diversity, but also may result in novel 15 insights into phage biology and functioning. Unfortunately, the study of soil viromes lags far 16 behind any other ecological model system, due to the heterogeneous soil matrix that rises 17 major technical difficulties in the extraction process. Resolving these technical challenges and 18 establishing a standardized extraction protocol is therefore a fundamental prerequisite for 19 replicable results and comparative virome studies. 20 Results 21We here report the optimization of protocols for extraction of bacteriophage DNA from soil 22 preceding metagenomic analysis such that the protocol can equally be harnessed for phage 23 isolation. As an optimization strategy, soil samples were spiked with a viral community 24 consisting of phages from different families (10 6 PFU/g soil): Listeria phage ΦA511 (Myovirus), 25Staphylococcus phage Φ2638AΔLCR (Siphovirus), and Escherichia phage ΦT7 (Podovirus). 26The efficacy of bacteriophage (i) elution, (ii) filtration, (iii) concentration, and (iv) DNA extraction 27 methods was tested. Successful extraction routes were selected based on spiked phage 28 recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes 29 were then extracted with the optimized methods and shotgun sequenced. Our approach 30 yielded sufficient amounts of inhibitor-free viral DNA for non-amplification dependent 31 sequencing and low 16S rRNA gene contamination levels (≤ 0.2 ‰). Compared to previously 32 published protocols, the number of bacterial read contamination was decreased by 65 %. In 33 addition, 468 novel circularized soil phage genomes in size up to 235 kb were obtained from 34 over 29,000 manually identified viral contigs, promising the discovery of a large, previously 35 inaccessible viral diversity. 36Conclusion 37 3 We have shown a dramatically enhanced extraction of the soil phage community by protocol 38 optimization that has proven robustness in both a culture-depended as well as through 39 metaviromic analysis. Our huge data set of manually curated soil viral contigs roughly doubles 40 the amount of currently available soil virome data, and provide insights into the yet largely 41 undescribed soil viral sequence space. 42 Keywords 43Bacteriophage extraction, phage metaviromes, soil samples, bacterial contamination 44 4 6 contigs. This data set roughly doubles the amount of today's available viral contigs from soil, 101 and opens the door for discovery of a very large soil viral diversity. 102 Results 103 Protocol optimization strategy 104The selective criteria for optimized extraction routes relied on three major parameters: (i) phage 105 yield at each step of the extraction route, (ii) reduction of bacterial DNA contamination levels 106 and (iii) bias...

IntroductionTuberculosis (TB) is an infectious disease caused by the group of bacterial pathogens Mycobacterium tuberculosis complex (MTBC) and is one of the leading causes of death worldwide. Timely diagnosis and treatment of drug-resistant TB is a key pillar of WHO’s strategy to combat global TB. The time required to carry out drug susceptibility testing (DST) for MTBC via the classic culture method is in the range of weeks and such delays have a detrimental effect on treatment outcomes. Given that molecular testing is in the range of hours to 1 or 2 days its value in treating drug resistant TB cannot be overstated. When developing such tests, one wants to optimize each step so that tests are successful even when confronted with samples that have a low MTBC load or contain large amounts of host DNA. This could improve the performance of the popular rapid molecular tests, especially for samples with mycobacterial loads close to the limits of detection. Where optimizations could have a more significant impact is for tests based on targeted next generation sequencing (tNGS) which typically require higher quantities of DNA. This would be significant as tNGS can provide more comprehensive drug resistance profiles than the relatively limited resistance information provided by rapid tests. In this work we endeavor to optimize pre-treatment and extraction steps for molecular testing.MethodsWe begin by choosing the best DNA extraction device by comparing the amount of DNA extracted by five commonly used devices from identical samples. Following this, the effect that decontamination and human DNA depletion have on extraction efficiency is explored.ResultsThe best results were achieved (i.e., the lowest Ct values) when neither decontamination nor human DNA depletion were used. As expected, in all tested scenarios the addition of decontamination to our workflow substantially reduced the yield of DNA extracted. This illustrates that the standard TB laboratory practice of applying decontamination, although being vital for culture-based testing, can negatively impact the performance of molecular testing. As a complement to the above experiments, we also considered the best Mycobacterium tuberculosis DNA storage method to optimize molecular testing carried out in the near- to medium-term. Comparing Ct values following three-month storage at 4 °C and at −20 °C and showed little difference between the two.DiscussionIn summary, for molecular diagnostics aimed at mycobacteria this work highlights the importance of choosing the right DNA extraction device, indicates that decontamination causes significant loss of mycobacterial DNA, and shows that samples preserved for further molecular testing can be stored at 4 °C, just as well at −20 °C. Under our experimental settings, human DNA depletion gave no significant improvement in Ct values for the detection of MTBC.