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Page 4 of 12 Jones et al. Microbiome Res Rep 2024;3:24 https://dx.doi.org/10.20517/mrr.2023.78

RESULTS
In silico screening for putative bacteriocin gene clusters
[30]
This study screened 181 bacterial urobiome isolates cultured from catheterised urine samples.
Catheterised urine samples reduced the risk of cross-contamination from surrounding microbiomes
[32]
(urethra, skin, vagina, etc.), and, as such, represent true urobiome/bladder isolates . The initial screening
using BAGEL4 and antiSMASH7 resulted in the identification of 263 AOIs [Supplementary Table 1].
BAGEL4 identifies the presence of AOIs within the genomes; however, this does not necessarily translate
into functional peptide production for reasons including mutations, regulation, or target specificity.

In total, 263 AOIs were identified from 97 isolates across 35 genera, with 54 of the isolates predicted to
produce more than one putative bacteriocin [Supplementary Table 1]. Further analysis revealed that 83 of
the AOIs lacked a core structural peptide sequence. Of these, 72 of the identified bacteriocin operons
contained the full complement of accessory genes necessary for bacteriocin production but appeared to lack
the required core structural peptides. Thirty-six strains encoded sactipeptide gene clusters with no core
peptide, and 16 strains encoded bottromycin but again lacked the structural core peptide from 25 different
genera including Actinomyces, Aerococcus, Bacillus, Fingoldia, Gordonia, Staphylococcus, Klebsiella, Leclercia,
Morganella and Pseudomonas. A possible explanation for this is that the BAGEL4 database may simply
not contain the sequence homologues of the core peptides . It has also been hypothesised that
[33]
bacteriocin production can be spontaneously acquired in the microbiome by horizontal gene transfer and
can be lost by deletion of biosynthetic genes as bacteriocin production is metabolically costly [18,34] . Similarly,
20 putative helveticin J peptides predicted by BAGEL4 were excluded from further analysis due to the lack
of a core peptide. It is also noteworthy that, in some studies, helveticin J peptides are no longer classed as
bacteriocins and are considered a distinct group of antimicrobials (called bacteriolysins) .
[28]

Of the remaining 180 putative bacteriocin AOIs, 100 were determined to be lacking the key associated genes
for bacteriocin production and, as such, were eliminated [Figure 1]. This resulted in 32 remaining isolates
with 80 PBGCs that contained a structural core peptide and the associated accessory genes [Supplementary
Table 2]. While they were removed based on the parameters set for this study, we do accept the possibility
that these gene products may work in conjunction with other novel bacteriocins/bacteriocin-related genes
[33]
encoded elsewhere on the genome . Of the 80 remaining bacteriocins, 72% were identified as class II,
13.75% as class III, 8.75% as class I, and 5% as unclassified bacteriocins.

Further analysis of PBGCs of particular interest
Based on BAGEL4, BLASTP, and EBI EMBOSS Needle analysis of the urobiome isolates, 53 putative
bacteriocin hits were determined to be potentially novel [Supplementary Table 2]. Novelty in this case is
taken as a core peptide with two or more amino acid differences compared to its closest characterised
homologue , or a 100% identity to previously uncharacterised bacteriocin with no other closely related
[10]
characterised homologues. Three bacterial strains, Lactobacillus gasseri UMB0099, Streptococcus
macedonicus UMB0733, and Proteus mirabilis UMB0315, were chosen for further analysis [Table 1,
Figures 2 and 3].

Analysis of selected novel PBGCs identified among Lactobacillus species
Lactobacillus strains were identified most often (54%) when analysing urobiome isolates for novel
bacteriocin production. Lactobacillus is one of the most common bacterial species isolated from the
urobiome, particularly in women [13,28,35] , and is thought to play a protective role within the urobiome .
[36]
Lactobacillus species are well-characterised bacteriocin producers and have been highlighted for their
[22]
potential applications in medicine, veterinary medicine, and the food industry as effective alternatives to