Data Availability StatementThe metagenomic data collection generated was deposited to BioProject (PRJNA590575). samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the U0126-EtOH distributor cultures, enhancing the recovery of bacteria up to 6 logs. WG5, with the exception of blood (13.5%). Lysis plaques were not observed in U0126-EtOH distributor any of the other bacterial species tested (and WG5 (%)*34 (56.7)7(13.5)25 (50)27(48.2)21(39.6)114 (42.1)With phages showing infectivity and observed by TEM (%)27 (45)6 (11.5)14 (28)8 (14.3)12 (22.6)77 (28.4)WG5, with and were not observed. Later, confirmation of the phage particles obtained from samples showing positive lysis on was conducted by Transmission Electron Microscopy U0126-EtOH distributor (TEM). TEM observation of phages directly isolated from the samples was performed in those samples containing more than 107C108 phage particles/mL, the minimal required for TEM visualization13. Below this concentration no phage particles will be observed. When phages were not so abundant and therefore not observed by direct analysis, they were then recovered from the lysis plaques generated on did not allow observation by TEM. The lowest phage detection rate by TEM was in blood samples (11.5%), while serum, a sample expected to produce similar results, showed higher percentages in both analyses (infectivity and TEM). On average, infectious phages were observed in 42.1% of the samples and in the 28.4% of them it was possible to visualize phage particles by TEM (Table?1). (the most frequent), and morphological type were observed (Table?1). Open in a separate window Physique 1 Electron micrographs of phages from ascetic fluid, blood, serum, cerebrospinal fluid (CSF) and urine. (and (NT) structures compatible with phage capsids but not showing a tail. Bar 100?nm. Virome analysis Four pools of urine samples and one pool of ascitic fluid (AF) samples allowed the recovery of viral DNA in sufficient quantity and purity to generate the libraries. Before the capsids were broken, the samples were tested for 16S rRNA genes, and unfavorable results confirmed the absence of non-packaged DNA and the effectiveness of the protocol18. Analysis of the virome showed a great number of unclassified sequences, that was greater in the AF and in two urine pool samples (Table?2), but the abundance of unclassified sequences did not correlate with the number of phages detected. The viromes confirmed the presence of bacteriophages in the samples and revealed coincidences with phages infecting different bacterial genera (Table?2; Fig.?2). Even if the identification of phage sequences by Kraken suggests a possible bacterial DDX16 host, this cannot be confirmed only by sequence comparison with the databases. Nevertheless, all samples showed sequences of phages coincident with phages infecting and (1), (2), (1), (1), (1), (22), (1), (1), (1), (2), (1), crAssphage** (1)1417 urines50078697 (13973.96 Mbp)170329 (60.57 Mbp)15491 (9.09%)154838 (90.91%)64(1), (1), (3), (7) (1), (5), (1), (1), (1), (36), (1), (4), (1), crAssphage (1)1521 urines3516412 (999.81 Mbp)35005 (14.99 Mbp)17188 (49.10%)17817 (50.90%)47(1), (1), (2), (4), (1), (1), (1), (29), (1), (1), (5), crAssphage (1)1615 urines4867915 (1359.94 Mbp)30101 (20.00 Mbp)22117 (73.48%)7984 (26.52%)44(1) (1), (3), (1), (1), (4), (30), (2), crAssphage (1)176 urines14093699 (3501.02 Mbp)131414 (51.45 Mbp)5362 (4.08%)126052 (95.92%)29(1), (1), (1), (3), (18), (4), crAssphage (1) Open in a separate window *Number of different phages detected in each virome that show homology with sequences in the databases of phages infecting the bacterial host described. **crAssphage is usually a particular type of human-specific phage infecting genera. Open in a separate window Physique 2 Identification of the 16S rRNA gene sequences found in the virome of a pool of ascitic fluids, and four pools of urine samples. On the.