Microbiological Research 259 (2022) 127010

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

The urinary microbiome and biological therapeutics: Novel therapies for

urinary tract infections
Ciara Kenneally, Craig P. Murphy, Roy D. Sleator, Eamonn P. Culligan *
Department of Biological Sciences, Munster Technological University, Bishopstown, Cork T12 P928, Ireland

A R T I C L E I N F O A B S T R A C T

Keywords: The discovery of microbial communities in the urinary tract (the urobiome) has fundamentally altered the
Antibiotic Resistance previous doctrine regarding urine sterility and associated urinary disorders. Recent advances in culturing and
Asymptomatic Bacteriuria culture-independent DNA sequencing technologies have characterised the resident microbial community in the
Biotherapeutics
urobiome, and has, in turn, demonstrated how community imbalances potentially contribute to infection and
Urobiome
Uropathogenic E. coli
disease. As we enter a post-antibiotic era, the effectiveness of standard antimicrobial treatments against multi-
UTI drug resistant (MDR) uropathogens is vastly diminished. Preliminary research is accumulating surrounding
microbiome-based therapies, and their potential as non-antibiotic therapeutics. In this context, the urobiome is
significantly underexplored, and knowledge regarding the fundamental role of its constituents is lacking. Herein,
we review the current state of the art concerning the urobiome; specifically, how it impacts health and disease
states, in the context of urinary tract infections (UTIs). Furthermore, we discuss the development of novel bio­
logical therapeutics that may have the potential to provide significant advancements in UTI therapy, with a
particular focus on bacterial interference, probiotics, antimicrobial peptides, bacteriocins, and bacteriophage.

1. Introduction
The human body is host to a diverse community of microorganisms,
establishing symbiotic relationships with one another and their host
(Sleator, 2010). Extensive studies examining the human microbiome
have generated an unprecedented understanding of the microorganisms
that colonise both the human body and its external environment (Cull­
igan et al., 2014). In turn, much has been learned about the metabolic
and immune function of the microbiome, and the interrelated dynamics
between a microorganism’s relative abundance (Rizzetto et al., 2018;
Visconti et al., 2019). However, not all parts of the body were deemed
suitable for initial exploration.
The urinary tract was not included in early microbiome research, as
it was originally thought to be a sterile environment (Magistro and Stief,
2019). Urine samples containing bacteria were believed to be contam­
inated by the external environment, due to difficulties associated with
sampling and microbiological analysis (Ainsworth, 2017). However,
over the last decade the existence of a urinary microbiome (urobiome)
has been acknowledged, and shown to consist of a complex microbial
community (Wolfe et al., 2012; Whiteside et al., 2015; Antunes-Lopes
et al., 2020; Jones et al., 2021). Although studies are relatively recent,
the existence of a ‘core’ urobiome has been hypothesized, with periodic
shifts in the microbial community leading to infection (Lewis et al.,
2013; Ammitzbøll et al., 2021; Zandbergen et al., 2021). Indeed, one of
the most common bacterial infections in humans affects the urinary tract
(Guglietta, 2017). Globally, urinary tract infections (UTIs) account for
~150 million infections annually, and ~40% of all hospital-acquired
infections (Nicolle, 2008; Totsika et al., 2011). Women are dispropor­
tionally affected by UTIs with > 50% women developing at least one UTI
over the course of their lifetime (Foxman, 2014). Risk factors among
different age groups tend to vary, and overall women develop more UTIs
than males due to anatomical and physiological differences, such as a
shorter urethra and hormone fluctuations (Abelson et al., 2018). A
higher frequency of sexual intercourse is a risk factor for younger co­
horts and lower oestrogen levels predisposes postmenopausal females to
developing UTIs (Alperin et al., 2019; Jung and Brubaker, 2020; Price
et al., 2020b). Furthermore, one-in-three women in the United States
will have received antibiotic therapy for their first UTI before the age of
twenty-four (Foxman et al., 2000). Uropathogenic reservoirs often pre­
vail within the urinary tract, and the surrounding vaginal and gut
microbiomes, causing recurrent UTIs in up to 30% of individuals within
6-months of the initial infection (Vagios et al., 2020). This high

* Correspondence to: Department of Biological Sciences, Munster Technological University, Cork, Ireland.
E-mail address: eamonn.culligan@mtu.ie (E.P. Culligan).

https://doi.org/10.1016/j.micres.2022.127010
Received 6 January 2022; Received in revised form 15 March 2022; Accepted 16 March 2022
Available online 20 March 2022
0944-5013/© 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
C. Kenneally et al.
prevalence of recurrent infections results in significant healthcare costs.
In the United States alone, the annual economic burden of UTIs is esti­
mated to exceed 3.5 billion dollars (Flores-Mireles et al., 2015).
Financial costs are not the only burden associated with UTIs, as
antimicrobial resistance (AMR) among uropathogens continues to
evolve at an alarming rate (Hrbacek et al., 2020). Conventional anti­
biotic therapy causes long-term alterations in the urobiome, chronic
recurring cystitis, and the development of MDR pathogens; often
relating to prolonged episodes of infection (Raz, 2001; Kostakioti et al.,
2012). Despite this, broad-spectrum antibiotics remain the first-line
treatment for UTIs (Bonkat et al., 2020). Consequently, approximately
80% of uropathogens now display resistance to at least two antibiotics
(Ahmed et al., 2019). Thus, MDR UTIs now represent a real and sub­
stantial health threat (Sihra et al., 2018).
At present, functional knowledge regarding the mechanistic rela­
tionship between the urobiome and the host remains obscure (Jones-­
Freeman et al., 2021). However, resident microbial communities in
other body sites are known to play critical roles in maintaining host
health (Krishnan et al., 2017; Dimitri-Pinheiro et al., 2020; Manor et al.,
2020). Further application of systems biology principles and ‘omics’
technologies could unravel the functional capabilities of the urobiome,
thus, benefitting diagnostic and therapeutic approaches in urogynae­
cology (Bullman et al., 2012). This will likely be of value in clinical
practice, potentially identifying diagnostic biomarkers for targeting
during clinical investigations (Claesson et al., 2017). Moreover, micro­
organisms derived from such communal ecosystems could play a central
role as natural biological tools, and, when engineered, show promising
potential as novel therapeutic interventions (Sleator and Hill, 2008a,
2008b). Herein, we review the current knowledge of live bio­
therapeutics within the urinary tract, and their prospective beneficial
contributions to urinary tract health. We also discuss emerging methods
and technologies for sequencing the urobiome and the insight such tools
have given in enabling us to interpret alterations between healthy and
infected microbial communities of the urinary tract.
2. Description of the methodology
A literature search was performed using SCOPUS, PubMed, and
Google Scholar from inception to September 2021. Keywords included
the following terms: ‘Microbiome’ OR ‘Asymptomatic Bacteriuria’ OR
‘Urobiome’ AND (‘UTI’ OR ‘Interstitial Cystitis’ OR ‘Pyelonephritis’ OR
‘Antimicrobial Resistance’ OR ‘Uropathogens’) OR ’UTI’ AND
(‘Asymptomatic Bacteriuria’ OR ‘Probiotics’ OR ‘Bacteriophage’ OR
‘Antimicrobial Peptide’ OR ‘Bacteriocin’). Types of articles retrieved
included original research articles, animal model studies, randomised
controlled trials, clinical trials, and reviews. The initial search identified
766 papers, all of which were reviewed by title and abstract, and
screened for eligibility. Articles excluded were those without an ab­
stract, not written in English, and clinical studies lacking information of
urine sampling techniques.
3. The urinary microbiome
3.1. Urine culturing
The establishment of the Human Microbiome Project (HMP) has led
to an increased understanding of the role of microbes in human health
and disease (Human Microbiome Project Consortium, 2012). Due to the,
now dated, belief that the urinary tract was a sterile environment, it was
not included in the HMP as a body site to characterise (Hilt et al., 2014).
Significant limitations are associated with standard urine culture
methods in terms of ability to isolate bacteria, however, optimised
culturing and culture-independent DNA sequencing technologies have
revealed the low-biomass microbial community within the urinary tract
(Wolfe and Brubaker, 2015).
Traditional urine culturing employs 5% sheep blood agar and
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Microbiological Research 259 (2022) 127010
MacConkey agar, aerobically incubated at 35 ◦C (Hilt et al., 2014).
When diagnosing UTIs, such culture techniques are limited in their
detection abilities, favouring the growth of fastidious, aerobic,
Gram-negative uropathogens (Wolfe et al., 2012). The diagnostic
“gold-standard” relies on a quantitative threshold of ≥ 105 CFU/ml for
adequate diagnosis of bacteriuria causing UTIs (Nicolle et al., 2005).
However, the validity of such detection methods is questionable as an
estimated 90% false-negative rate has been reported for standard urine
culturing (Hilt et al., 2014).
Enhanced quantitative urine culture (EQUC) could improve detec­
tion of clinically relevant urinary microbes, while additionally
advancing our understanding of commensal and pathogenic microor­
ganisms among the urobiome (Price et al., 2016). Whilst the
expanded-spectrum EQUC protocol involves plating increased urine
volumes and additional growth media with differing oxygen or carbon
dioxide enriched atmospheres, a streamlined adaptation could provide
sufficient information to clinicians when assessing the treatment re­
quirements for uropathogens (Hilt et al., 2014; Pearce et al., 2014; Price
et al., 2016). Blood agar, colistin-nalidixic acid agar, and MacConkey
agar are utilised in the streamlined EQUC protocol and incubated in 5%
CO2, diversifying the detection of urotypes, the dominant taxa in urine
samples (Price et al., 2016). Urotypes describe community profiles
within the urobiome, typically dominated in relative abundance by one
organism (Gottschick et al., 2017). Additional advancements in DNA
sequencing have increased the identification of urotypes based on the
relative abundance of operational taxonomic units (OTUs) in cluster
analysis (Pearce et al., 2015; Ammitzbøll et al., 2021). Only 33% of
known uropathogens were detected with standard urine culturing, while
the streamlined EQUC protocol identified 84% (Price et al., 2016).
Contrasting the ≥ 105 CFU/ml threshold, the EQUC method can
generally detect between 102-105 CFU/ml of urine, useful in the context
of a low biomass community, such as the urinary tract microbiota
(Mueller et al., 2017).
3.2. Culture-independent sequencing of the urinary microbiome
On average, it is estimated that the EQUC protocol captures
approximately 72% of the total microbial genera present in the uro­
biome (Thomas-White et al., 2018). The dawn of the ‘omics’ era has
provided increased detection capabilities, as DNA sequencing technol­
ogies can assess the collective microbial composition inhabiting a spe­
cific environment (Franzosa et al., 2015). Large-scale metagenomic
projects have not only led to the detection of novel genes and species,
but have also expanded the functional characterisation of the human
microbiome in terms of metabolic pathways and physiological adapta­
tions required for survival (Sberro et al., 2019; Dekaboruah et al., 2020).
Two sequencing strategies are commonly employed when assessing
the microbiome: 16 S ribosomal RNA (rRNA) gene amplicon sequencing
and whole-genome sequencing (WGS) (Human Microbiome Project
Consortium, 2012; Thomas-White et al., 2016). Combined with urine
culturing, 16 S rRNA gene amplicon sequencing has been more
commonly employed in studies characterising the urobiome (Karstens
et al., 2021). Nine hypervariable regions (V1-V9) exist in the 16 S rRNA
gene (Van de Peer et al., 1996). When assessing the phylogenetic
relatedness, the hypervariable regions are utilised as they contain spe­
cific polymorphisms (Brubaker and Wolfe, 2015; Thomas-White et al.,
2016). Deciding the chosen hypervariable region targeted during 16 S
rRNA gene sequencing is important, as different hypervariable regions
might limit the taxonomic identification, causing microbial taxa to be
either under- or over-represented (Mizrahi-man et al., 2013). Both the
V1 and V6 regions, for example, have reportedly been inadequate when
compared to other hypervariable regions, demonstrating suboptimal
performance in phylogenetic analysis due to limited region length and
lower average sequence identities (Yu and Morrison, 2004; Grasp­
euntner et al., 2018). A direct comparison evaluating how different
hypervariable regions perform when capturing the composition of the
C. Kenneally et al.
urobiome has not yet been conducted (Karstens et al., 2018). WGS can
comprehensively characterise complete genomes by sequencing the
entire DNA sequence (Neugent et al., 2020). Although WGS is a more
expensive technique, it generates sequence reads to a higher resolution
and usually offers a more detailed taxonomic and functional profile
(Franzosa et al., 2015). Mutations in the FimH adhesin, for example,
were significantly correlated to the B2 phylogroup of uropathogenic
E. coli (UPEC) isolates, when performing a comparison on faecal and
urinary isolates, using WGS (Nielsen et al., 2017). WGS has also enabled
the identification of anaerobic members of the Actinobacteria, Firmi­
cutes, and Bacteroidetes, which were not previously identified using
culture-only methods (Thomas-White et al., 2018).
A study by Moustafa et al. (2018) assessed the complementarity of
the sequencing methods. 16 S rRNA gene sequencing exhibited greater
sensitivity when detecting low-abundance DNA in voided urine samples
(Moustafa et al., 2018). Nevertheless, taxonomic profiling and func­
tional resolution is limited with 16 S rRNA gene sequencing, whilst WGS
provides an increasingly specific functional and taxonomic classification
(Jovel et al., 2016). However, WGS can be subject to amplifying con­
taminants attained from the human host, generally from the skin or
vaginal microbiomes, possibly as a result of urine sampling methods
(Moustafa et al., 2018). A limitation associated with both sequencing
methods is that non-viable, ruptured bacteria can be detected (Karstens
et al., 2018; Boers et al., 2019). It is hypothesised that the identification
of non-viable bacteria is still beneficial as they once played a role within
the microbiome, potentially providing increased functional knowledge
(Aragón et al., 2018). However, this could lead to misinterpretation
when assessing the urobiome’s extant state. Therefore, a combined use
of EQUC and DNA sequencing technologies is recommended, where
EQUC will identify live microorganisms (Karstens et al., 2018).
Both techniques have identified as-yet-uncultured organisms from
urinary samples (Fouts et al., 2012; Garretto et al., 2018; Cummings
et al., 2020). Urine samples analysed with 16 S rRNA gene sequencing
contained the Atopobium genus, which was not identified using culturing
methods (Aragón et al., 2018). Non-prokaryotic organisms are also more
frequently detected using WGS compared to culture-only protocols,
namely the eukaryotic yeast species Candida (Moustafa et al., 2018).
Atopobium and Candida have previously been identified within the
vaginal microbiome, where increased abundance correlates with the
onset of vaginal infection. Atopobium has been identified in samples
derived from individuals suffering with bacterial vaginosis (Ferris et al.,
2004) and Candida species have been associated with sexually trans­
mitted diseases and vaginal thrush (Moustafa et al., 2018). Most studies
surrounding the urobiome thus far have been limited in size, focused on
taxonomic profiling with 16 S rRNA gene sequencing, and thus knowl­
edge regarding the functionality of members of the urobiome remains
limited (Jones-Freeman et al., 2021).
3.3. Profiling the urinary microbiome
Urine is typically comprised of bacteria from four phyla: Firmicutes,
Bacteroidetes, Proteobacteria, and Actinobacteria (Karstens et al.,
2016). A ‘core’ urobiome is being elucidated, however, most studies to
date, analysing the urinary tract have focused on the female urobiome,
due to the wide range of urological disorders affecting women
(Schneeweiss et al., 2016; Brubaker and Wolfe, 2017). Discrepancies
also persist among urine sampling techniques, whereby voided urine is
often subject to post-urethral contamination (Wolfe et al., 2012; Bru­
baker et al., 2021). Additionally, a recent study has also unveiled dif­
ferences between bacteria detected in urine samples and bladder
mucosal tissue samples (Mansour et al., 2020). Capturing tissue samples
along with urine would give further insight into various bladder dis­
eases, however sampling techniques are more invasive (Mansour et al.,
2020). Nevertheless, differences between the male and female urobiome
have been reported (Gottschick et al., 2017).
Both the male and female urobiome are dominated by the phylum
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Microbiological Research 259 (2022) 127010
Firmicutes, while Actinobacteria and Bacteroidetes are lacking in the
male urobiome (Nelson et al., 2010; Lewis et al., 2013; Karstens et al.,
2016). Overall, the male urobiome is depicted as a less complex envi­
ronment, typically distinguished from the female urobiome based on the
relative abundance of different genera (Fouts et al., 2012; Ceprnja et al.,
2021). Corynebacterium has been identified as the dominant genus of the
male urobiome, a genus commonly present within the skin microbiome
(Fouts et al., 2012). Other bacterial genera commonly identified in both
the male and female urobiome include Lactobacillus, Streptococcus, and
Staphylococcus (Gottschick et al., 2017). Generally, the urobiome of
males in the > 70-years cohort appears to be more diverse than that of
females, potentially correlated with the increased risk of prostate, kid­
ney, and bladder diseases (Wojciuk et al., 2019). For example, Propio­
nibacterium acnes strains similar to those present in the male skin
microbiome had a higher abundance in prostate cancer patients, while
Enterobacteriaceae and Actinobaculum urinale uropathogens were prev­
alent in benign tumours (Mak et al., 2013; Shrestha et al., 2018). A
cluster of proinflammatory bacteria including Streptococcus anginosus,
Anaerococcus spp., Varibaculum cambriense, and Propionimicrobium lym­
phophilum have also been linked to urological infections and malignant
tissues in males (Domann et al., 2003; Williams, 2015; Shrestha et al.,
2018). Microorganisms present in the male urobiome seem to resemble
those present in the skin microbiome, whilst the female urobiome ex­
hibits a commonality with the vaginal microbiome (Fouts et al., 2012;
Lewis et al., 2013).
Urotypes in the female urobiome are commonly dominated by
Shigella, Lactobacillus, Enterococcus, Gardnerella, Prevotella, Sneathia,
Escherichia, and Streptococcus (Gottschick et al., 2017; Price et al.,
2020a). Lactobacillus is identified as the dominant genus in a healthy
female urobiome (Hilt et al., 2014; Pearce et al., 2015). Lactobacillus is
also abundant in the vaginal microbiome, with certain species contrib­
uting to a healthy vaginal state, when present at adequate levels,
maintaining a lower pH and protecting against pathogens (Marrazzo,
2011). A similar function has been proposed for Lactobacillus in the
urobiome, correlating with a decrease of the genus in postmenopausal
women and the subsequent increase of recurrent UTIs (rUTIs) (Staple­
ton, 2016). Inhibition of uropathogen growth has also been shown in the
presence of lactic acid and hydrogen peroxide; by-products of Lactoba­
cillus rhamnosus and Lactobacillus reuteri metabolism (Cadieux et al.,
2009). Low-to-moderate levels of Lactobacillus in premenopausal women
also correlates with urinary incontinence, indicating an imbalance from
the healthy microbiome (Komesu et al., 2018). Whilst taxonomic
profiling of the urobiome has advanced significantly, it is still not known
how temporal factors, such as sex hormones, menstrual cycle, and sexual
intercourse contribute in altering the microbial composition (Gümüş
et al., 2019; Price et al., 2020b). Early studies focused on the temporal
dynamics of the female urobiome have determined a likeness to the skin
microbiome during menstruation, and an increase in Staphylococcus and
Streptococcus linked to sexual intercourse (Price et al., 2020b).
Gardnerella is another genus frequently isolated from urine samples,
more specifically Gardnerella vaginalis (Pearce et al., 2014, 2015; Kars­
tens et al., 2016). Gardnerella is also reportedly increased in the vaginal
microbiome post sexual intercourse (Gajer et al., 2012). G. vaginalis is
abundantly present in females suffering with bacterial vaginosis, and is
associated with an increased risk of women developing a UTI (Ravel
et al., 2011; Morrill et al., 2020). Gilbert and colleagues (2017), using a
mouse model, determined that G. vaginalis can function as a driving
factor causing the re-emergence of UPEC from latent intracellular res­
ervoirs. This suggests that G. vaginalis overgrowth is a plausible trigger
for pyelonephritis in women, causing increasingly serious manifesta­
tions, resulting in severe kidney infections and epithelial exfoliation
(Gilbert et al., 2017; O’Brien et al., 2020). High abundance of Gard­
nerella in the female urobiome is associated with a subsequent decrease
in Lactobacillus species, however a dysbiotic relationship has not been
verified due to the presence of Gardnerella in healthy individuals (Pearce
et al., 2014). Large-scale, longitudinal studies will be required to further
C. Kenneally et al. Microbiological Research 259 (2022) 127010

evaluate the implications of alterations in genus-level abundances and to
identify promising biomarkers.
3.4. Modulation of the urinary microbiome during UTI
3.4.1. Recurrent UTIs
UPEC dominates UTI pathogenesis, being detected in 80% of clinical
urine samples tested using standard culture techniques (Klein and
Hultgren, 2020). However, the detection of emerging uropathogens is
reducing the heretofore E. coli-centric depiction of UTIs (Price et al.,
2018; Hochstedler et al., 2021). Several species, including Pseudomonas
aeruginosa, Enterobacter aerogenes, Klebsiella pneumoniae, Aerococcus
urinae, Proteus mirabilis, and Streptococcus anginosus are increasingly
identified by modern DNA sequencing methods as causative agents of
UTIs (Price et al., 2016, 2018). A comparison of the urobiome in a
healthy state and during UTI is presented in Fig. 1.
UPEC pathogenesis remains the primary concern for recurring in­
fections. Reinfection occurs in approximately 25%− 30% of women
within 6 months of a preceding UTI (Vagios et al., 2020). The pathobi­
ology of rUTIs is still not fully understood, and longitudinal meta­
genomic analysis is lacking in rUTI patients. One proposed mechanism is
that reinfection evolves from UPEC populating the gastrointestinal tract
or urogenital epithelial reservoirs (Nielsen et al., 2014a). UPEC isolates
colonise the periurethral area, subsequently moving to the urethra and
ascending through to the bladder during UTI pathogenesis (Flores-Mir­
eles et al., 2015; Terlizzi et al., 2017). After adherence to urogenital
epithelial cells, UPEC can invade the cytosol causing aggregation and
formation of intracellular bacterial communities (IBCs), typically within
a biofilm-like matrix (Hirakawa et al., 2019). The presence of IBCs have
been detected in individuals suffering from acute cystitis and rUTIs
(Rosen et al., 2007; Robino et al., 2013). Schreiber and team (2017)
detected same-strain rUTIs in 12 out of 18 rUTI incidences, likely
resulting from latent reservoirs. Internal uropathogen reservoirs appear
to persist in the bladder and vaginal walls of postmenopausal females;
increasing the risk of rUTIs (De Nisco et al., 2019).
UPEC strains and filamentous uropathogens involved in the forma­
tion of IBCs are highly pathogenic, contributing to re-infection, resis­
tance to antimicrobials, and infection chronicity (Hannan et al., 2012;
McLellan and Hunstad, 2016). Phylogenetic analysis typically divides
E. coli strains into four main groups (A, B1, B2, and D), however updated
PCR-based methods recognise three additional groups (designated C, E,
and F) (Clermont et al., 2000). Phylogroup B2 and D are associated with
virulent UPEC strains (Schreiber et al., 2017; Biggel et al., 2020).
Notably, UPEC clones originating in the gut derived from phylogroup B2
and D possess a higher level of multidrug resistance (Nielsen et al.,
2014a). UPEC presence in faeces demonstrates that the urogenital and
gut microbiomes are not mutually exclusive. Forde et al. (2019) evalu­
ated the population dynamics of a virulent E. coli ST131 lineage from
voided urine and faecal samples, over a 5-year period, to understand
rUTI aetiology. An individual harboured an intestinal reservoir of
UTI-inducing E. coli ST131 variants causing reoccurrence over a 5-year
period, further linking a relationship between the gut and urobiome
(Forde et al., 2019). Interestingly, the ST131 clone conferred resistance
against more antibiotics initially, losing multiple resistance plasmids by
the end the fifth year (Forde et al., 2019). The authors suggested that the
loss of resistance plasmids was a mechanism of adaption to fluctuating
antibiotic regimens (Forde et al., 2019). This warrants a requirement for
temporal and long-term analysis of the evolution of bacteriuria in IBC’s
and the impact on the urobiome. Persistent community alterations
appear to correlate with an increase in latent reservoirs amongst the
elderly, and despite antibiotic therapy, > 60% of rUTIs are attributed to
the original uropathogenic strain (Russo et al., 1995; Thänert et al.,
2019; Nielsen et al., 2021).
3.4.2. Polymicrobial UTIs
Standard urine culture relies on the belief that UTIs are generally
monomicrobial infections (Smelov et al., 2016). This has led to the
concept of ‘single-species UTIs’ in clinical laboratories, deeming

Fig. 1. Comparison of the urinary microbiome during a healthy state and UTI. (A) Most bacteria, such as Prevotella and Shigella, are commonly found in the
urobiomes of both healthy males and females. Lactobacillus and Corynebacterium species dominate the urobiomes of females and males, respectively. (B) Overview
of risk factors predisposing UTI pathogenesis. Immunocompromised, elderly, indwelling devices, and urogenital tract infections or previous antibiotic use greatly
increase the risk of infection. (C) UTIs are associated with increased abundance of common uropathogens such as E. coli, Enterococcus and Staphylococcus and a
subsequent decrease in Lactobacillus (arrow). Gardnerella species are also frequently associated with infection.

4
C. Kenneally et al.
multiple colony morphologies as contamination (Kline and Lewis,
2016). Despite a lack of studies surrounding polymicrobial UTIs, it is
hypothesised that such infections are more common than previously
assumed (Fourcade et al., 2015). Approximately 20% of women with
symptomatic UTIs result in culture-negative urine samples (Ferry et al.,
2007). Culture-negative is a misleading term as it includes samples with
polymicrobial growth, and pathogens present at < 105 CFU/ml (Kline
and Lewis, 2016). One study reported that 42% of women presented
with polymicrobial infections, characterised by the presence of at least
one Gram-positive and one Gram-negative bacterium (Kline and Lewis,
2016; Price et al., 2018). It is also estimated that 33% of UTIs among the
elderly are polymicrobial (Croxall et al., 2011). Polymicrobial UTIs are
frequently described among the immunocompromised and individuals
with indwelling catheters (Kline and Lewis, 2016). Werneburg and
co-authors (2020) detected polymicrobial uropathogenic biofilms on
catheters as early as 7-hours post installation, which further progressed
as indwelling time increased resulting in biofilms on the proximal and
distal portions by 26 days on average. As a result, these infections either
go untreated or are subject to inappropriate antibiotic interventions. The
damaging effects of this have also been noted by Werneburg et al. (2020)
whereby four out of ten sequence samples were noted to confer
beta-lactam, quinolone, aminoglycoside, and tetracycline resistance
genes.
Detrimental effects of antibiotics on the genitourinary microbiome
prompting the onset of polymicrobial UTIs and rUTIs have previously
been proposed. Discrepancies were demonstrated in an earlier study by
Rani et al. (2017) analysing the bladder microbiome of kidney trans­
plant patients treated with prophylactic trimethoprim-sulfamethoxazole
(TMP-SMX) via metagenomic sequencing. Two opportunistic pathogens,
Enterococcus faecalis and E. coli, both showed increased abundance when
sequenced after treatment, whilst a subsequent decrease in general mi­
crobial diversity was observed (Rani et al., 2017). Interestingly,
E. faecalis and UPEC have previously been reported to cause poly­
microbial infections within the urinary tract and surgical wounds
(Keogh et al., 2016). A synergistic relationship has been described be­
tween the two pathogens, upon which L-ornithine is secreted by
E. faecalis and subsequently stimulates E. coli’s iron uptake system,
efeUOB complex, in iron-limiting environments; thus, E. coli growth and
biofilm formation are increased (Keogh et al., 2016). E. coli has also
previously exhibited synergism with the yeast Candida albicans, simul­
taneously increasing one-another’s growth, possibly an aetiological
factor in fungal UTIs (De Brucker et al., 2015). However, not all inter­
species interactions are beneficial to pathogens. For instance,
P. aeruginosa displays dominance over E. coli, causing decreased growth,
likely due to secretion of virulence factors such as N-acyl-L-homoserine
lactones (Qin et al., 2009; Cerqueira et al., 2013). Generally, there is a
lack of comprehensive studies surrounding polymicrobial relationships
within the urobiome, thus, interpretations of microbial interplay largely
remain obscure (Azevedo et al., 2017).
3.4.3. Polymicrobial Biofilms
Biofilm formation is a common attribute associated with poly­
microbial communities, resulting from the assemblage of a collection of
organisms enclosed within an extracellular polymeric substance matrix
(Penesyan et al., 2020). Microbial culture and culture-independent ex­
aminations of catheter-associated UTIs (CAUTI) have determined that
biofilms contaminating urinary catheters are usually polymicrobial in
nature (Frank et al., 2009; Galván et al., 2016). Holá et al. (2010) re­
ported similar observations when only 12.5% of CAUTI were mono­
microbial. However, their findings relied on culture techniques, and as
such, it is likely that metagenomic methods could lead to the reported
12.5% being further reduced. Organisms previously associated with
polymicrobial infections identified on urinary catheters include
E. faecalis, P. mirabilis, and K. pneumoniae (Frank et al., 2009; Fourcade
et al., 2015; Armbruster et al., 2017; Werneburg et al., 2020). The un­
derlying mechanisms of most interspecies interactions within the
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Microbiological Research 259 (2022) 127010
urogenital tract are yet to be elucidated. Proposed explanations behind
such microbial interplay include metabolic signalling interactions that
are often accompanied by upregulation of virulence factors, reduced
wound healing, and altered antibiotic susceptibility (Pastar et al., 2013;
De Vos et al., 2017; Trizna et al., 2020).
Accumulating evidence suggests that polymicrobial interactions
heighten antimicrobial resistance, ultimately by hindering antibiotic
efficacy (Orazi and O’Toole, 2020). In turn, biofilm-mediated resistance
enables polymicrobial communities to withstand high concentrations of
antimicrobial agents (Gaston et al., 2020). The extracellular matrix also
offers additional resistance mechanisms, containing β-lactamases and
degrading antibiotics upon entry, before they can reach their target
bacterium (Høiby et al., 2010). For instance, E. coli isolates obtained
from polymicrobial UTIs appear more invasive and display increased
resistance to antibiotics such as ciprofloxacin and trimethoprim (Croxall
et al., 2011). Depending on interspecies interactions, mixed-species
biofilms either enhance resistance or exhibit tolerance towards antimi­
crobial therapy; a phenomenon termed “recalcitrance” (Lebeaux et al.,
2014). Enterococcus and Proteus species regularly co-colonise the urinary
tract and indwelling catheters, and are notorious biofilm producers
(Fourcade et al., 2015; Armbruster et al., 2017). Alarmingly, P. mirabilis
and E. faecalis biofilms severely limit antimicrobial agents, conferring
resistance to β-lactam antibiotics, aminoglycosides, fluoroquinolones,
and carbapenems (Ch’ng et al., 2019; Gaston et al., 2020). Additionally,
urease production is primarily associated with P. mirabilis, causing urine
obstruction and crystalline biofilm formation on catheters (Soto, 2014;
Pelling et al., 2019). The crystalline layer also provides heighted pro­
tection to multi-species biofilms on catheters, providing resistance
against pre-coated antimicrobial agents (Goh et al., 2017). The conse­
quences of such interspecies interactions can lead to metabolite
cross-feeding and altered susceptibility towards antibiotics (Short et al.,
2014; Bottery et al., 2021), contributing to enhanced disease progres­
sion and adverse patient outcomes compared to monomicrobial
infections.
4. Antibiotic resistance
UTIs have been disproportionately affected by antibiotic misuse
(O’Grady et al., 2019), increasing the prevalence of MDR uropathogens
(Mazzariol et al., 2017). Currently, the clinical guidelines that define
UTIs lends itself to equivocal interpretations, often leading to unnec­
essary administration of broad-spectrum antibiotics (Finucane, 2017).
Surveillance data accumulated by The Global Prevalence of Infections in
Urology (GPIU) suggests that approximately 56% of patients in urology
units were prescribed antibiotics, of which 26% were prescribed for
proven UTIs, while 21% were for suspected UTIs (Johansen et al., 2006;
Wagenlehner et al., 2016). Ultimately, the consequences for over­
prescribing antibiotics for non-confirmed UTIs correlates to a significant
increase in MDR uropathogens (Cek et al., 2014). Consequentially,
guidelines for antibiotic prescriptions for UTIs have been updated to
reflect the rise in resistance. Empiric fluoroquinolone treatment is now
only recommended if the frequency of local resistance in E. coli is ≤ 10%
and if the patient has not received fluoroquinolone treatment within the
last 6 months (Gupta et al., 2011; Bonkat et al., 2020).
Long-term modifications to microbiome constituents are also
impacted by antibiotic regimens. Antibiotics commonly employed for
UTIs are said to impact the gut microbiome, decreasing diversity and
abundance of commensals (Elvers et al., 2020). Studies investigating the
influence of antibiotics on the urobiome are limited, yet still concerning.
Antibiotics have been noted to significantly contribute to communal
variations in the urobiome (Gottschick et al., 2017; Mulder et al., 2019).
Gottschick et al. (2017) evaluated a two-fold increase in Lactobacillus
iners post metronidazole treatment, and the relative abundance of
Gardnerella vaginalis, Atopobium vaginae and Sneathia amni was signifi­
cantly decreased. Although Lactobacillus are typically acknowledged for
their health-optimizing characteristics, L. iners is controversial relating
C. Kenneally et al. Microbiological Research 259 (2022) 127010

to significant abundance in diseases such as bacterial vaginosis (Petrova
et al., 2017). When investigating the effect of several antibiotics on the
urobiome, Mulder et al. (2019) noted a subsequent increase in E. coli and
Parabacteroides abundance in voided urine samples, while Finegoldia and
Lactobacillus were decreased. Lack of oestrogen in postmenopausal
women also decreases Lactobacillus abundance in the urobiome whilst
simultaneously enhancing the diversity of uropathogens, increasing the
prevalence of infection and frequency of antibiotic treatment (Alperin
et al., 2019). Allowing such women access to oestrogen therapy can aid
in postmenopausal issues, including increasing Lactobacillus prolifera­
tion within the urogenital microbiome (Gliniewicz et al., 2019). How­
ever, it is yet to be associated with the complete prevention of UTIs (Raz,
2001; Thomas-White et al., 2020). Thus, developing new therapeutics
for UTIs is warranted as generally the archetypal resident urobiome
microbial population cannot be restored after responding to antibiotic
therapy (Gottschick et al., 2017).
prone to rUTIs, where one group was subject to antibiotic therapy while
participants in the control group were not. The frequency of reoccur­
rence was significantly higher in the group taking antibiotics (46.8%)
compared to the control group (13.1%), and presence of ABU was sug­
gested to play a protective role (Cai et al., 2012). Cai et al. (2012),
(2015) also concluded that E. coli isolates from the antibiotic group
displayed significantly higher resistance against amoxicillin–clavulanic
acid, ciprofloxacin and TMP-SMX. Hence, treating ABU with antibiotic
therapy is likely detrimental and might, in fact, damage the urobiome.
Thus, the European Association of Urology (EAU) and American Uro­
logical Association (AUA) recommend avoid treating ABU unless
examining a particular subgroup; pregnant women and individuals un­
dergoing surgery in which the bladder mucosa will be breached (Anger
et al., 2019; Bonkat et al., 2020).
6. Potential BioTherapeutics

5. Asymptomatic bacteriuria versus symptomatic UTI Table 1 summarises the biotherapeutics that have potential to
modulate the urobiome, while an overview of the mechanisms of action
Asymptomatic bacteriuria (ABU) is defined as two consecutive pos­ is presented in Fig. 2.
itive urine specimens (≥105 CFU/ml) isolated from an individual lack­
ing symptoms attributable to a UTI (Nicolle et al., 2005). E. coli
6.1. Bacterial Interference
predominantly cause ABU (Roos et al., 2006a; Dobrindt et al., 2016).
ABU prevalence increases with age, occurring in 20% of women by the
E. coli 83972 is currently the most well characterised strain associ­
age of 80%, and 3.6%− 19% in community-dwelling elderly men after
ated with ABU (Darouiche et al., 2005; Köves et al., 2014; Stork et al.,
60 years (Nicolle, 2003; Roos et al., 2006c). The frequency increases to
2018). E. coli 83972 was first isolated from a young girl, who harboured
25–50% in women, and 15–40% in men in long-term care facilities;
the strain for ~3 years without displaying symptoms of infection
likely due to complications such as impaired functional status and uri­
(Lindberg et al., 1975; Andersson et al., 1991). Although E. coli 83972
nary incontinence, ultimately leading to long-term catheterisation
resembles commensal E. coli, studies indicate that the strain evolved
(Nicolle, 1999; Leihof et al., 2021). However, a misunderstanding sur­
from a uropathogenic progenitor (Klemm et al., 2006). Indeed, the strain
rounds ABU, and as to whether such strains should be considered as
has been linked to the E. coli B2 phylogenetic group, the predominant
commensal organisms or potential pathogens due to their phylogenetic
phylogroup associated with UPEC strains (Roos et al., 2006b). Virulence
relatedness to uropathogens causing symptomatic infection (Finucane,
factors associated with UPEC strains that are present in E. coli 83972
2017).
include the expression of colibactin and cytotoxic necrotizing factor 1
Underlying and confounding medical issues predispose institution­
(CNF-1) (Stork et al., 2018). However, mutations differentiating the
alised adults to developing a higher frequency of ABU strains (Ajayi and
83972 ABU strain from E. coli strains that cause symptomatic infection,
Radhakrishnan, 2016). Simultaneously, this increases antibiotic overuse
including lack of motility, and the absence functional P, type 1, and F1C
as UTIs are diagnosed based on the presence of ≥ 105 CFU/ml bacteri­
fimbriae due to fim deletions and papG point mutations, are factors in
uria in urine samples (Nicolle et al., 2005). Thus, long-term care facil­
attenuating its virulence (Klemm et al., 2006; Roos et al., 2006b;
ities have become hotspots for the spread of resistant uropathogens
Zdziarski et al., 2008, 2010).
(Rowe and Juthani-Mehta, 2014). ABU strains present in the urobiome
Bacterial interference can be established with E. coli 83972 as the
typically present in a state of commensalism; not causing symptoms in
strain outcompetes UPEC isolates, decreasing UTI risk. E. coli 83972
their host. Cai et al. (2012) evaluated the influence of ABU in women
colonisation, as opposed to UPEC, has been previously documented

Table 1
Biotherapeutics displaying antimicrobial activity against uropathogens.
BiotherapeuticName Type Mode of Action Clinical Evidence Reference

E. coli 83972 Asymptomatic Bacterial interference.Competition for nutrients. Phase 2 – significant reduction of rUTIs Sundén, Bonkat et al.,
Bacteriuria Biofilm interference. Bacteriocin production. (N = 20). No active phase 3. Approved for use (2010, 2020)
in LUTD by the EAU.
Pyophage Bacteriophage Virus infecting bacteria. Phase 3 – non-inferior to antibiotics (N = 113). Leitner et al. (2021a)
RCT commencing in spinal cord patients. Leitner et al. (2021b)
Lactin-V (Lactobacillus Probiotic Lactic acid and hydrogen peroxide production. Phase 2 – rUTI incidence rate reduced to 15% Stapleton et al. (2011)
crispatus) Blocks adherence to uroepithelial cells. (N = 50). No active phase 3.
L. rhamnosus GG Probiotic Lactic acid and hydrogen peroxide production. Phase 2 – 1 or 2 doses safe in NLUTD and SCI Groah et al. (2019)
Downregulates NF-κB, P, and Type 1 fimbriae. patients (N = 80). No active phase 3.
Biofilm interference.
Mutaflor (E. coli Nissle Probiotic Microcin production. Competition for iron using In Vitro – no significant UPEC reduction.Phase Pradhan and Weiss
1917) iron uptake systems. 4 in active development for UTIs children. (2020)Tapiainen
(2020)
Faecal Microbiota Donor Stool Bacterial interference. Preliminary clinical study – significant Ghani et al. (2021)
Transplantation Transplant reduction of rUTIs (N = 90).
Cecropin A Antimicrobial Membrane permeabilization.Inhibits efflux pump In Vivo – significant UPEC biofilm reduction in Fenner, (2020)Kalsy
Peptide and nucleic acids interactions. Biofilm interference. G. mellonella model. et al. (2020)
Colicin E2 Bacteriocin Endonucleolytic degradation of DNA. Preliminary catheter trials – complete Trautner et al. (2005)
inhibition of susceptible E. coli.

*rUTIs: recurrent urinary tract infection, LUTD: lower urinary tract dysfunction, EAU: European Association of Urology, RCT: randomised controlled trial, NLUTD:
neurogenic lower urinary tract dysfunction, SCI: spinal cord injury.

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C. Kenneally et al. Microbiological Research 259 (2022) 127010

Fig. 2. Mechanisms of action of biotherapeutics against uropathogens including probiotics, bacteriocins and antimicrobial peptides. (A) Probiotic bacteria are
competitive microorganisms fighting for nutrients and urothelium adherence, and interfering with biofilms. Production of lactic acid regulates the pH of the
environment, providing an unsubstantial medium for uropathogen survival. (B) Bacteriocins act as toxins against competing bacteria, degrading their DNA, inhibiting
peptidoglycan synthesis, and/or forming pores in membranes. Small molecules, amino acids, and ions leak from the cell, inducing cell death. (C) Efflux pumps can be
inhibited by additional antimicrobial peptides, affecting transport of toxic compounds and bacterial metabolites.

7
C. Kenneally et al.
through pre-inoculation of urinary catheters and deliberate establish­
ment in biofilms (Trautner et al., 2003; Ferrières et al., 2007). The
clinical effectiveness of the strain has been investigated in small scale
studies, where introduction of E. coli 83972 into neurogenic bladders
reduced the reoccurrence of UTIs (Sundén et al., 2010). There is limited
knowledge regarding how the ABU outcompetes UPEC, with suggestions
including outcompeting for nutrients and attachment sites, gene
acquisition, and inhibiting biofilm production (Darouiche and Hull,
2012; Beatson et al., 2015; Stork et al., 2018). Competition for attach­
ment sites has been disproved, as E. coli 83972 lacks the fimbriae
required for adherence (Hull et al., 2002; Roos et al., 2006b; Zdziarski
et al., 2010). Despite confusion surrounding the exact mechanism of
action, clinical use of E. coli 83972 is endorsed for deliberate bladder
colonisation by the European Urology Guidelines (Dobrindt et al., 2016;
Bonkat et al., 2020). Aside from the prototype 83972 strain, additional
ABU strains are not as well characterised. A study by Stork et al. (2018)
showed that two ABU isolates (strain 9 and 106) displayed an increased
competitive advantage against two model strains, E. coli 83972 and the
UPEC 536 strain. In total, nine ABU strains were evaluated, however as
they were only competing against two model strains, their performance
against other uropathogenic strains remains unelucidated (Stork et al.,
2018). Both strain 9 and 106 appeared to contain additional fitness
factors, such as bacteriocin expression and siderophore aerobactin sys­
tems, potentially contributing to their interference (Stork et al., 2018).
Deliberate colonisation with E. coli 83972 in humans is regarded as safe,
thus larger studies with additional ABU strains could lead to further
development of novel biotherapeutics (Köves et al., 2014;
Jones-Freeman et al., 2021).
6.2. Microbiota transplantation
Faecal microbiota transplantation (FMT) involves transferring the
microbial community in a suspension of minimally manipulated stool
from a healthy donor into the intestinal lumen of a patient, restoring a
healthy microbial composition (Culligan and Sleator, 2016). Recently,
FMT therapy has repopulated commensals in the urobiome, which has
been associated with a reduction of rUTIs. In a small case study per­
formed by Tariq et al. (2017), a significant decrease of rUTI incidences
was detected after FMT treatment for recurrent Clostridium difficile
infection (rCDI). Susceptibility to antibiotics subsequently increased,
and abundance of E. coli and Klebsiella uropathogens reduced (Tariq
et al., 2017). Biehl et al. (2018) detected similar reductions in Entero­
bacteriaceae from an abundance of 8.3–0.5% 84 days after FMT in a
50-year-old female, while Lactobacillaceae abundance increased from
~0.5% to > 50%. Similarly, in a 93-year-old female, FMT reduced
symptomatic rUTIs and eradicated rCDIs (Aira et al., 2021). The authors
reasoned the vast reduction in symptomatic infections resulted from the
reduction of Enterobacteriaceae in the gut from 74% pre-FMT to 0.07%
post-FMT (Aira et al., 2021). FMT has shown positive results in a slightly
larger cohort of 9 patients in rUTI reduction, with only one episode of
recurrence from ESBL-producing strains (Ghani et al., 2021). Pre­
liminary case studies to date have investigated FMT in few participants
and large-scale studies would benefit the confirmation of this therapy.
FMT’s renowned success in reducing rCDI has opened the question as
to whether microbial transplantation from other niche’s would be an
effective therapy. Vaginal microbiota transplantation (VMT) has not
been trialled in individuals suffering with rUTIs, but has been presented
as a potentially viable treatment for bacterial vaginosis (Lev-Sagie et al.,
2019). This was the first reported VMT performed in humans, at the time
of writing, and yielded in long-term remission of bacterial vaginosis in 4
out of the 5 participants (Lev-Sagie et al., 2019). Additionally, a Lacto­
bacillus-dominated microbiome was restored after VMT, re-forming
healthy vaginal microbiome configurations (Lev-Sagie et al., 2019).
Given the interrelatedness of the vaginal microbiome with the uro­
biome, it could be explored whether this treatment strategy could be
viable for rUTIs and other urological diseases. On a similar note, we have
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Microbiological Research 259 (2022) 127010
previously proposed the concept of urinary microbiota transplantation
(UMT) to target rUTIs (Jones et al., 2021). Enhancing this trans­
plantation therapy with health-associated strains such as ABU or
Lactobacillus could perhaps identify a novel biological therapy for rUTIs.
6.3. Phage therapy
Whilst relatively less is known about the urobiome when compared
to other human microbomes, even fewer studies have focused on the
viral constituents of the urinary tract, otherwise known as the urinary
virome (Santiago-Rodriguez, 2018). Although shifts within the uro­
biome community are significantly affected by health status, viral con­
stituents appear to be unaltered (Santiago-Rodriguez et al., 2015).
Presently, metagenomic analysis of the urinary virome has primarily
focused on eukaryotic viruses, where Human Papilloma Virus (HPV) is
the most abundant (Shigehara et al., 2010; Rani et al., 2016). Pre­
liminary metagenomic analysis has revealed that novel phage sequences
in the urinary virome exceed eukaryotic viruses (Miller-Ensminger et al.,
2018). For instance, Santiago-Rodriguez and colleagues (2015) have
determined that 27% of viral sequences in urine were homologous to
known viruses, out of which 99% represented bacteriophage genes.
However, this only represents a fraction of phages present, meaning a
large unexplored viral community exists in the urinary tract (Miller-­
Ensminger et al., 2018; Garretto et al., 2019).
Currently, studies surrounding phage therapy for UTIs are typically
comprised of lytic proteins, phage cocktails, or phages used synergisti­
cally with antibiotics (Malik et al., 2020). Lytic phages naturally pro­
duce two types of lytic proteins targeting lysis of the cell wall; endolysins
and virion-associated peptidoglycan hydrolases (PGH), also referred to
as virion associated lysins (VALs) (Gutiérrez et al., 2018). Additionally,
lytic phages are reported as potential therapies for MDR uropathogens
(Galtier et al., 2016; Bagińska et al., 2021). Sybesma and co-authors
(2016) investigated the in vitro susceptibility of nine urine derived
K. pneumoniae isolates to lytic phages. Notably, significant activity was
displayed by one phage, v_BR–KpS10, as 100% of K. pneumoniae isolates
were lysed (Sybesma et al., 2016). More recently, the lytic phage
VB_ecoS-Golestan has displayed activity against troubling UPEC strains,
inhibiting 56% of MDR UPEC isolates (Yazdi et al., 2020). Restricting
factors, such as limited host range and phage-resistant mutants, do limit
the efficacy of single phage therapies for UPEC treatment (Malik et al.,
2020).
The high specificity phages display in their single-phage form not
only restricts their ability to treat polymicrobial infections, but also in­
creases the risk of phage resistant bacteria evolving (Gurney et al., 2017;
Wright et al., 2019). Thus, phages in combination with another anti­
microbial, and phage cocktails are typically preferred. Polyphage and
combination therapies are particularly advantageous when treating
polymicrobial infections and biofilms (Malik et al., 2020). For instance,
a phage cocktail targeting MDR Acinetobacter baumannii led to > 90%
reduction in biofilm biomass when combined with TMP-SMX and cip­
rofloxacin (Grygorcewicz et al., 2021). The synergistic effect typically
results from the antibiotics’ mode of action, inhibiting dihydrofolate
reductase (DHFR), DNA topoisomerase II, and DNA topoisomerase IV,
and provoking faster phage expansion and cell lysis (Chegini et al.,
2021). Synergism has also been reported between antipseudomonal
phages and a genetically modified ABU strain, E. coli HU2117, a deriv­
ative of E. coli 83972 with an 800-base pair deletion in chromosomal
papG (Liao et al., 2012). As a result of the chromosomal gene deletion, P
fimbriae, virulence factors associated with bacteremia and pyelone­
phritis (Hull et al., 2002), are no longer produced by E. coli HU2117.
Liao and co-authors (2012) reported an almost 4-log reduction in
P. aeruginosa biofilm formation on catheters when E. coli HU2117 and
the phage (ΦE2005-A) were combined, whilst neither displayed signif­
icant inhibition alone. However, the effectiveness of antibiotic-phage
combinations are highly dependent on the chosen antibiotic and bac­
terial pathogen, as demonstrated by the antagonistic activity seen
C. Kenneally et al.
between a MDR A. baumannii phage cocktail and colistin (Grygorcewicz
et al., 2021). Phage binding and reproduction were limited when com­
bined with colistin due to the degradation of the cell membrane (Dan­
is-Wlodarczyk et al., 2016).
Pyophage, a commercial phage produced by the Eliava Institute
(Tbilisi, Georgia), has been investigated in initial clinical studies for the
treatment of UTI-causing uropathogens when administered (Leitner
et al., 2017). Pyophage is a phage cocktail that typically targets E. coli,
Pseudomonas, Proteus, Staphylococcus and Streptococcus strains causing
wound or skin infections (Abedon et al., 2011). In a primary in vitro
clinical study, the commercial Pyophage was adapted after a series of
liquid titrations to determine the lowest concentration affective against
bacteria (Ujmajuridze et al., 2018). Subsequently, sensitivity of bacteria
to a 4-fold adapted Pyophage increased from 41% to 75% (Ujmajuridze
et al., 2018). Furthermore, the adapted phage on a catheter caused a
1–5 log reduction in bacterial titers in six out of nine participants,
whereby E. coli was detected in four participants, and both Streptococcus
spp. and Enterococcus spp. were only present in two participants post
treatment (Ujmajuridze et al., 2018). Recently, in a randomized
controlled clinical trial consisting of 113 patients, Leitner and team
(2021b) reported that the efficacy and safety of the adapted intravesical
Pyophage is non-inferior to antibiotic treatment for UTIs. Intravesical
administration is a method of prophylaxis where a drug is administered
directly into a patient’s bladder using a catheter (GuhaSarkar and
Banerjee, 2010). Additionally, bladder irrigation with the phage cocktail
appeared superior in males undergoing transurethral resection of the
prostate (TURP) (Leitner et al., 2021b). Leitner and colleagues (2021a)
have also hypothesized the potential use of phage treatment for in­
dividuals with spinal cord injuries evidently suffering with UTIs and
were to commence clinical trials in 2020. Therefore, whilst phage
research shows promising results when eradicating uropathogens,
further clinical studies are required to validate their effectiveness.
6.4. Probiotics
6.4.1. Gram-positive
Probiotics are widely defined as “live microorganisms that, when
administered in adequate amounts, confer a health benefit on the host”
(Guarner et al., 2012). Antimicrobial compounds, such as organic acids,
bacteriocins, biosurfactants, and hydrogen peroxide, enhance the
antagonistic properties of probiotic bacteria aiding in the prevention of
various diseases/disorders (Darouiche and Hull, 2012). Lactic acid from
Lactobacillus SCS, in concentrations above 30 mM, exhibits potent
antibacterial properties by eliminating UPEC (Cadieux et al., 2009).
However, Cadieux et al. (2009) indicated that Lactobacillus, which
produce lactic acid, could also transform it into a carbon source bene­
ficial to UPEC, explaining the cause of infections when Lactobacillus
colonisation still persists. Hydrogen peroxide production can adversely
affect UPEC adhesion factors through the upregulation of stress proteins
OmpA and OmpX, inducing membrane distress (Stancik et al., 2002;
Wang, 2002; Kim et al., 2010). Upregulation of such proteins could lead
to the reduction of biofilm formation in enteric and genitourinary en­
vironments (Li et al., 2018).
Typically, probiotics are consumed orally, however alternative de­
livery routes are possible, including vaginally (Wolff et al., 2019). As
latent UPEC reservoirs can colonise the genital tract (Brannon et al.,
2020), vaginal probiotics could potentially decrease UPEC reservoirs
and subsequently decrease rUTIs. Commensal lactobacilli regulate the
vaginal microbiome, working to maintain homeostasis by reducing
pathogen abundance (Marrazzo, 2011). Lactobacillus crispatus is one of
the most effective strains in maintaining a healthy microbiome (Nardini
et al., 2016). L. crispatus CTV-05 is the active probiotic strain in Lactin-V,
a live biotherapeutic product in clinical trials for treating bacterial
vaginosis (Cohen et al., 2020). When assessing Lactin-V’s potential in
treating uropathogens only 15% of the 50 treated individuals developed
rUTIs, while the placebo cohort had a 27% rUTI incidence rate
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Microbiological Research 259 (2022) 127010
(Stapleton et al., 2011). Additionally, L. rhamnosus is one of the most
frequently studied Lactobacillus strains (Petrova et al., 2018; Pino et al.,
2019). It promotes the downregulation of UPEC virulence factors
including nuclear factor-κB (NF-κB) eukaryotic transcription factors, P
and type 1 fimbriae, and also interferes with biofilms (Cadieux et al.,
2009; McMillan et al., 2011; Karlsson et al., 2012; Petrova et al., 2016).
Randomised controlled trials were conducted with 252 postmenopausal
females, evaluating the efficacy of an oral probiotic containing
L. rhamnosus GR-1 and L. reuteri RC-14 against antibiotic therapy
(Beerepoot et al., 2012). When compared against TMP-SMX the pro­
biotic did not meet the non-inferiority criteria for UTI inhibition, how­
ever, presence of antibiotic resistant uropathogens were significantly
reduced within the probiotic cohort (Beerepoot et al., 2012). Vaginal
administration of a L. rhamnosus GR-1 and L. reuteri RC-14 probiotic has
previously produced promising results; reducing UTI reoccurrence from
47% to 21% when used alongside antibiotics (N = 41) (Reid et al.,
1992). Additionally, Lactobacillus acidophilus’ inhibitory effects against
E. coli displayed the highest inhibitory potential (60%), warranting
further investigation into the strain (Velraeds et al., 1998; Vagios et al.,
2020). A new era of probiotic administration could be on the horizon, as
intravesical delivery of lactobacilli have shown promising potential in
individuals with neurogenic lower urinary tract dysfunction (Groah
et al., 2019). Currently, intravesical gentamicin irrigation is deemed
effective for individuals suffering with rUTIs when all other systemic
treatments are unsuccessful (Dray and Clemens, 2017). When adminis­
tered in one or two doses, a L. rhamnosus GG intravesical probiotic was
found to be safe and well tolerated in participants with cloudy or
foul-smelling urine (Groah et al., 2019). It was hypothesized that a
community shift in the urobiome could result from intravesical pro­
biotics, however the study is still in preliminary stages (Forster et al.,
2019). Despite the success of in vitro studies with oral/vaginal
L. rhamnosus, the strain has not yet shown significant reduction in UTI
occurrence, or UPEC reduction (Forster et al., 2019; Groah et al., 2019).
Although this is the first human trial utilizing intravesical probiotics,
Lactobacillus casei demonstrated significant antimicrobial activity
against chronic UTIs in murine models (Asahara et al., 2001).
6.4.2. Gram-negative
E. coli Nissle was one of the first strains utilized as a probiotic, and
has been commercialised as the main active component of the probiotic
Mutaflor for the treatment of gastrointestinal disorders (Schultz, 2008;
Scaldaferri et al., 2016). E. coli Nissle 1917 has also shown promise as a
prophylactic agent against urological infections (Storm et al., 2011).
Genomic analysis between the avirulent E. coli strains Nissle 1917 and
83972, and a virulent uropathogen E. coli CFT073 revealed a close ge­
netic relationship between the three strains, suggesting that the strains
likely originated from a common ancestor (Hancock et al., 2010b; Vej­
borg et al., 2010). Increased genetic similarity between Nissle 1917 and
CFT073 was identified due to the presence of a pathogenic island
PAI-CFT073-serU, predominantly identified in strains causing pyelone­
phritis (Vejborg et al., 2010). Additionally, expression of F1C fimbriae is
associated with Nissle 1917, contributing to the strain’s adherence
capability to different cells (Grozdanov et al., 2004; Kleta et al., 2014).
Expression of fitness factors contribute to E. coli Nissle’s 1917
antagonism, subsequently promoting the bacterium’s biofilm formation
(Hancock et al., 2010a; Scaldaferri et al., 2016). A ferric iron uptake
system comprising of the siderophore yersiniabactin, has previously
been identified in the strain, contributing to biofilm formation in
iron-limiting environments (Grozdanov et al., 2004). The fyuA gene,
encoding the yersiniabactin receptor, is one of the most upregulated
genes in biofilm-producing strains, including those causing UTIs (Han­
cock et al., 2008). E. coli Nissle 1917 contains an array of additional iron
uptake systems (enterobactin, aerobactin, ferric dicitrate transport sys­
tem, and the chu heme transport locus) facilitating survival through
heme import (Garcia et al., 2011; Scaldaferri et al., 2016). Expression of
two known catechol microcins, microcin M and microcin H47,
C. Kenneally et al.
represents yet another advantageous function of E. coli Nissle (Patzer
et al., 2003; Vassiliadis et al., 2010). Catechol microcins are post­
translational modified peptides characterized by a catechol-siderophore
moiety (Patzer et al., 2003; Thomas et al., 2004). Under iron-limited
conditions, such microcins utilize their catechol-siderophores contrib­
uting to their bacterial competitiveness by binding ferric iron (Thomas
et al., 2004; Vassiliadis et al., 2010). This mechanism has aided in their
antibacterial function against Enterobacteriaceae causing intestinal in­
fections (Schultz, 2008; Baquero et al., 2019). Despite E. coli Nissle’s
probiotic properties, the strain has not yet displayed significant reduc­
tion in UPEC, possibly due to antimicrobial defences in UPEC strains
(Pradhan and Weiss, 2020). However, E. coli Nissle 1917 has gained
increased attention as a model organism for the development of novel
therapeutics (Geldart et al., 2018; Praveschotinunt et al., 2019).
6.5. Antimicrobial peptides
Antimicrobial peptides are small polypeptide antimicrobials
(<10 kDa), part of the innate immune system, and secreted by host
tissues (Suda et al., 2017). As part of the host defence system, AMPs are
produced by the urothelium of the bladder, ureter, kidneys, and the
urethra in the urinary tract (Becknell et al., 2015). AMPs exert direct
antimicrobial activity, and can also function as immunomodulatory
compounds (O’Sullivan et al., 2020). Their net cationic charge, amphi­
pathicity, and secondary structure contribute to their ability to bind to
target cells and disrupt the lipid bilayers (Yeaman and Yount, 2003;
Ebenhan et al., 2014; Becknell et al., 2015). Along with the bio­
therapeutic potential of AMPs, they can also be eminent biomarkers as
expression levels can often correlate with infection (Gupta et al., 2018).
Several known antimicrobial peptides detected in the urinary tract have
been thoroughly evaluated to determine their function in both mice and
humans, with cathelicidin, human β-defensins, and human α-defensin 5
(HD5) appearing to be the most prevalent (Hiratsuka et al., 2000;
Chromek et al., 2006; Zasloff, 2007; Spencer et al., 2014).
Human cathelicidin (LL-37) is an α-helical AMP, primarily expressed
in circulating neutrophils, epithelial cells, and myeloid bone marrow
cells (Agerberth et al., 2000). Although evidence surrounding LL-37’s
protective role in the urinary tract is limited, some studies have reported
promising results (Spencer et al., 2014; Babikir et al., 2018; Gupta et al.,
2018). For example, when challenged with UPEC invasion, increased
expression of mRNA in the CAMP LL-37 gene, and subsequent increased
LL-37 secretion, was detected within minutes (Chromek et al., 2006).
Deletion of the murine LL-37 homologue, CRAMP, resulted in higher
UPEC infection rates as bladder colonisation increased (Ali et al., 2009).
Additionally, LL-37-resistant E. coli are increasingly present in more
invasive infections, such as pyelonephritis, and increased resistance is
displayed in urine and faecal samples of patients suffering with rUTIs as
opposed to healthy individuals (Chromek et al., 2006; Nielsen et al.,
2014b). LL-37 can suppress biofilm formation by inhibition of CsgA
polymerization, a subunit of curli fimbriae (Chromek, 2015). Presence
of excess curli fimbriae gives rise to resistance in UPEC strains, inhib­
iting LL-37 from disrupting the bacterial membrane (Kai-Larsen et al.,
2010; Luna-Pineda et al., 2019).
Defensins are biologically active peptides classified into α- and
β-defensins in humans, dependent on the sequence of cysteine residues
and disulphide bridges (Becknell and Spencer, 2016). Significant in­
creases in the presence of the HD5 have been associated with patients
suffering from pyelonephritis, and with patients undergoing urinary
diversion surgery (Townes et al., 2011; Spencer et al., 2012). The AMP’s
full potential is yet to be elucidated, however a recombinant HD5 has
been shown to exhibit bactericidal activity against a variety of uro­
pathogens (Wang et al., 2010). Tikhonov et al. (1997) also detected an
eight-fold increase in urinary α-defensin HNP-1 during chronic pyelo­
nephritis, correlating with an increase in cytokine IL-8 and leukocyte
levels, suggesting neutrophil recruitment (Tikhonov et al., 1997).
However, microbiological information regarding the HNPs relationship
10
Microbiological Research 259 (2022) 127010
to uropathogens is yet to be elucidated (Ali et al., 2009; Becknell et al.,
2015). β-defensins are more widely expressed in humans, and both
human β-defensin-1 (HBD-1) and human β-defensin-2 (HBD-2) have
been identified in the urinary tract (Becknell and Spencer, 2016).
Limited research has been conducted on HBD-2’s function in the urinary
tract, although the AMP has displayed activity against Enterococcus
faecalis (Kandaswamy et al., 2013). HBD-1 is predominantly expressed
by epithelial cells in the kidney, and its presence is increased three-fold
during acute pyelonephritis (Hiratsuka et al., 2000; Nielsen et al.,
2014b). Deficiency of mouse BD-1 did not alter UPEC pathogenesis in
vivo¸ while mouse BD-3 and BD-4 displayed bactericidal activity against
UPEC (Becknell et al., 2013). Interestingly, human β-defensins are
expressed in different levels in the genital tract throughout the men­
strual cycle (King et al., 2003; Ali et al., 2009), which could indicate a
correlation to the possible interconnectivity of the urinary and vaginal
microbiomes. Oestrogen supplementation has also been shown to
correlate with an increase in AMPs, including β-defensins and LL-37, in
the urinary tract (Lüthje and Brauner, 2016).
AMPs derived from insects (Manniello et al., 2021) and plants
(Monteiro et al., 2019), and antifungal peptides (Raman et al., 2016)
also represent potential treatments for human UTIs. Recently, cecropin
A (CecA) derived from the wax moth Galleria mellonella displayed ac­
tivity as a novel therapeutic against biofilm-forming UPEC strains (Kalsy
et al., 2020). This is achieved through a multi-target mechanism, where
the outer membrane is permeabilised, resistance-nodulation-division
(RND) efflux pumps are suppressed, and nucleic acid interactions are
inhibited, subsequently inhibiting biofilm formation (Kalsy et al., 2020).
A synergistic reaction between CecA and the antibiotic nalidixic acid
(NAL) was also observed, ultimately improving the entry of NAL into
cells, leading to a new approach to eradicate uropathogen biofilms
(Fenner, 2020).
6.6. Bacteriocins
Bacteriocins, a subcategory of AMPs, are ribosomally synthesised
peptides produced by bacteria to kill competing organisms, which are
usually closely related to the producer strain (Cotter et al., 2005). Bac­
teriocins produced by Gram-positive bacteria have been the focal point
of bacteriocin research, with many extensive reviews on the topic
(Mathur et al., 2017; Acedo et al., 2018; Soltani et al., 2020).
Gram-negative bacteria were not originally regarded as good candidates
for bacteriocin treatment, due to their outer lipopolysaccharide mem­
brane and subsequent reduced antibiotic efficacy (Egan et al., 2017).
However, the rise in MDR Gram-negative bacteria has led to a renewed
interest in alternative therapeutics, including Gram-negative
bacteriocins.
Gram-negative bacteriocins can be classified into two main groups,
colicins and microcins (Engevik and Versalovic, 2017). Colicins are
proteins with a high molecular weight (>20 kDa), while microcins are
lower molecular weight peptides of < 10 kDa (Rebuffat, 2011; Karpiński
and Szkaradkiewicz, 2016). Colicins are more extensively characterised,
and are synthesised by > 50% of E. coli strains, encoded by small
multi-copy plasmids, or large low-copy plasmids (Braun et al., 1994;
Gillor et al., 2004). Three main mechanisms are utilised by colicins
when damaging target cells; membrane permeabilization through
voltage-dependent channels, cellular nuclease degradation, and inhibi­
tion of peptidoglycan synthesis (Cascales et al., 2007; de Zamaroczy and
Chauleau, 2011). Historically, colicins have primarily been identified in
enteric strains (Garcia-Gutierrez et al., 2019). Studies regarding their
inhibitory potential against uropathogens are limited, but promising
(Budič et al., 2011; Roy and Riley, 2019).
Notably, Šmajs and co-authors (2010) reported a higher percentage
of colicin E1 plasmids (ColE1) in UPEC strains when compared to con­
trols, detected in 22% of strains versus 10%, respectively. Thus, ColE1
was suggested to be a potential virulence factor (Rijavec et al., 2007;
Šmajs et al., 2010). Conversely, certain colicins have displayed
C. Kenneally et al.
inhibitory action, reducing biofilm formation on urinary catheters.
Coating of colicin E2-producing E. coli K-12 on urinary catheters
exhibited complete antibacterial activity against susceptible isolates,
but was not effective against mutant E. coli displaying colicin resistance
(Trautner et al., 2005). Additionally, combining a lubricant with the
novel colicin SR4, creating a prophylactic, achieved the same antimi­
crobial activity against uropathogens as gentamicin, whilst using 20–30
times less drug (Roy and Riley, 2019). Another mechanism of imple­
menting bacteriocin activity against UPEC involves the introduction of
recombinant bacteria. For example, genetically engineering the pro­
biotic Lactobacillus brevis DT24 to express colicin E2 can increase the
strain’s antimicrobial capabilities against UPEC (Trivedi et al., 2014).
Employing this form of biotherapy could inhibit uropathogens while
subsequently benefitting the microbiome, however it has not yet been
evaluated in vivo.
Characteristically, colicins are expressed by more E. coli strains,
however microcin encoding determinants have been identified more
often in UTI causing strains (Šmajs et al., 2010). Microcins are heat
resistant antimicrobial peptides encoded by plasmids, or chromoso­
mally, and display a broad spectrum of activity, mostly against Entero­
bacteriaceae species (Lagos, 2013). Microcins achieve antimicrobial
action through the inhibition of bacterial enzymes, membrane per­
meabilization, and utilisation of membrane components (Rodríguez and
Laviña, 2003; Rebuffat, 2011). UTI-inducing UPEC are associated with
an increase in chromosomally encoded microcin H47 and microcin M
(Šmajs et al., 2010; Massip et al., 2020). Interestingly, when both of
these microcins are expressed in E. coli Nissle 1917 they confer a sig­
nificant competitive advantage against UPEC as they induce a previ­
ously mentioned iron uptake system (Patzer et al., 2003; Vassiliadis
et al., 2010). Massip and co-authors (2020) also identified microcin H47
and microcin M precursors more frequently in ABU strains than in UPEC.
Whether or not such ABU strains displayed a competitive advantage
against UPEC strains was not established. At the time of writing, no
naturally produced microcins have displayed significant reduction in
UPEC isolates. However, antimicrobial activity against both
Gram-positive and Gram-negative organisms has been analysed using
the recombinant hybrid bacteriocin Ent35-MccV, combining enterocin
CRL35 and microcin V (Acuña et al., 2012). Production of the hybrid
bacteriocin was achieved by the fusion of the structural portions of
enterocin CRL35 and microcin V, encoded by munA and cvaC respec­
tively, by asymmetric PCR (Acuña et al., 2012). Potent activity was
exhibited against various strains of E. coli, including five UPEC strains
(Acuña et al., 2012, 2015). UPEC230 was the only strain which dis­
played resistance against Ent35-MccV, likely due to the strain’s own
production of microcin V and immunity proteins. Bacteriocins are
notoriously unstable, however bioengineering is a common method of
improving stability whilst simultaneously expanding their antimicrobial
activity (Navarro et al., 2020; Soltani et al., 2020). Thus, implementing
bioengineering techniques could potentially produce a therapeutic
bacteriocin which could be administered intravesically on urinary
catheters (Roy and Riley, 2019) or within engineered probiotics (Trivedi
et al., 2014), for the inhibition of uropathogens.
7. Bioengineering
Bacteriocins are typically gene-encoded and thus are amenable to
bioengineering to alter the peptides structure with the aim of creating a
mutant peptide with enhanced function (Culligan and Sleator, 2017).
Modification of bacteriocins can be induced by mutating
bacteriocin-encoding genes or fusing genes together with subsequent
expression in an alternative host (Gillor et al., 2005; Cotter, 2012).
Nisin, a model bacteriocin, is extensively exploited for engineering
purposes resulting from the substantial understanding of the lantibiotic
structure (Field et al., 2010). Bioengineering strategies including alter­
ations of single amino acid residues, amino acid arrangement, and
site-saturation mutagenesis of individual hinge residues have
11
Microbiological Research 259 (2022) 127010
contributed to the production of bacteriocin derivatives with improved
functionality, solubility, stability, and activity (Kuipers et al., 1992; Rink
et al., 2007; Field et al., 2008; Cotter et al., 2013).
Typically, bacteriocins are more commonly employed against Gram-
positive bacteria, primarily due to the outer membrane of Gram-
negative bacteria decreasing their efficacy (Egan et al., 2017). Bioen­
gineering of the N-terminal domain and the ‘hinge’ region in nisin
through both site-directed and site-saturation mutagenesis have been
particularly advantageous, expanding its antimicrobial spectrum against
Gram-negative pathogens such as Shigella and Pseudomonas spp. (Yuan
et al., 2004; Field et al., 2010; Healy et al., 2013). Additionally, bio­
engineered nisin variants S29A and S29G exhibited increased potency
against Gram-negative species, including E. coli (Field et al., 2012).
Gram-negative bacteriocins also offer a degree of flexibility to en­
gineer new derivatives (Behrens et al., 2017). Pheromonicin, for
example, is a fusion product formed by the combination of a
channel-forming Colicin Ia, and its cognate immunity protein from
E. coli, with a pheromone-encoding gene (agrD) from Staphylococcus
aureus (Ji et al., 1995; Mayville et al., 1999). Pheromonocin exhibits
specific activity against S. aureus, and has shown increased performance
compared to penicillin in mouse models (Gillor et al., 2004). Genetic
fusion has also been exhibited in the construction of chimeras combining
S-type pyocins with colicins, which exhibited antimicrobial activity
against both E. coli and P. aeruginosa due to inhibition of lipopolysac­
charide synthesis and DNA breakdown (Kageyama et al., 1996). More
recently, a similar strategy was adapted to enable the secretion of a
chimeric bacteriocin from engineered E. coli. Replacing the N-terminal
and central domains of colicin E3 with those of pyocin S3 produced the
chimeric bacteriocin CoPy (Gupta et al., 2013). Subsequent tagging of
the flagellar secretion, FlgM, generated the antibacterial agent
FlgM-CoPy, designed to kill a wild-type P. aeruginosa after detection of a
quorum sensing molecule N-(3-oxododecanoyl) homoserine lactone
(3OC12HSL) (Gupta et al., 2013). Incorporation of non-canonical amino
acids into microcin J25’s peptide structure resulted in several variants
exhibiting antimicrobial activity similar to, or higher than, wild-type
microcin (Piscotta et al., 2015). Furthermore, combining chitosan
nanoparticles with microcin J25 formed a stable nano-antimicrobial,
CN-MccJ25, exhibiting bactericidal activity against both
Gram-negative and Gram-positive strains (Yu et al., 2021). Further
implementation of bioengineering strategies on antimicrobial com­
pounds could eventually be the key to creating novel therapeutics,
reducing the reliance on antibiotics for UTIs.
8. Future outlook
It is apparent that the relative abundance of bacterial communities
has a significant influence on urinary tract health, contributing to the
risk of diseases and infections (Burnett et al., 2021; Zandbergen et al.,
2021). However, a significant knowledge gap still exists. For instance,
although the synergistic activities between E. coli and E. faecalis have
been previously described (Keogh et al., 2016), overall the mechanistic
and functional relationships between microbes in the urobiome remains
undefined. Integrating advanced ‘omics’ technologies such as metab­
olomics and metatranscriptomics could further our insight into meta­
bolic signalling activities within the urobiome, and its interactions with
other microbial communities (Franzosa et al., 2015). Gaining this in­
formation could evaluate how potential treatment options such as nat­
ural UMT could be of benefit to the urobiome constituents or if
manipulation of urine needs to occur for this to be a viable future
therapy. Lactobacillus strains have received the most attention for
reducing E. coli abundance. As similar benefits have been seen from ABU
strains (Stork et al., 2018), it may be warranted to deepen our functional
knowledge of these strains, adding to the existing data surrounding
E. coli 83972. This could further our understanding of their metabolic
interactions with UPEC and could determine their unexplored potential
against other uropathogens. Additionally, whether or not these strains
C. Kenneally et al.
display similarity to E. coli Nissle 1917 as potential probiotic organisms
would be an avenue to investigate. If this is the case, ABU strains
eventually may be main active components in probiotics, possibly
treating urological disorders. Thus, initiating large-scale screening
projects to identify more ABU strains, and characterising them at a
molecular and genetic level could be particularly valuable in designing
novel therapies.
The efficacy of the biological therapeutics identified to date have not
currently met the non-inferiority criteria when compared to antibiotics
in clinical studies to treat UTIs. Nevertheless, there is a movement to
exploit such biological tools from inside the microbiome and improve
their antimicrobial efficacy, developing novel therapies. Lactobacillus
abundance in the vaginal microbiome does increase post oral adminis­
tration of probiotics, yet a significant increase has not been detected in
the urobiome (Vagios et al., 2020). Direct instillation of Lactobacillus
into the bladder of animal models have shown more positive results,
suggesting a more promising route of administration when treating UTIs
(Asahara et al., 2001). However, a healthcare professional would be
required for bladder installation, which could be both costly and labo­
rious if repeat visits are necessary. Data regarding the treatment of UTIs
using Lactobacillus-containing probiotics is inconsistent, as studies vary
heavily in terms of model systems, delivery routes, and probiotic agents
(Vagios et al., 2020). In turn, their true clinical potential remains un­
determined. Additionally, while bacteriocins also exhibit promising
potential, a significant gap remains in relation to studies focusing on
bacteriocins from Gram-negative bacteria. Bioinformatic tools have also
increased our ability to detect these antimicrobials. Approximately 2000
bacteriocin coding genes had been identified in Gram-negative bacteria
by 2017, including in opportunistic pathogens such as E. coli, K. pneu­
moniae and P. aeruginosa (Sharp et al., 2017). The transition from
genome mining to in vitro and in vivo studies is also significantly un­
derrepresented with bacteriocins from Gram-negative origins (Behrens
et al., 2017). In their natural form the efficacy of biological therapies is
often limited, relating to the low stability and solubility, and their strain
specificity (Riley et al., 2012; Meade et al., 2020). Nonetheless, their
antimicrobial potential against uropathogens warrants further explora­
tion, proving advantageous when targeting uropathogen growth and
biofilm formation.
Over the last decade, bioengineering strategies have advanced the
exploitation of biological tools to enhance their antimicrobial function,
stress tolerance, and solubility (Sheehan et al., 2007; Field et al., 2008;
Hayes et al., 2019). Other microbiome’s have appeared to be rich
sources of antimicrobial compounds (Mousa et al., 2017; O’Sullivan
et al., 2019; Lagenaur et al., 2021), suggesting that screening the uro­
biome might also unveil a potentially rich source of novel antimicro­
bials. Bacteriocins detected in other environments have displayed
enhanced activity when expressed in a recombinant engineered host
(O’Sullivan et al., 2021). Similar strategies could be applied to potential
bacteriocins in the urobiome in an attempt to enhance their activity.
Bioengineering Lactobacillus or ABU, for instance, as host expression
systems for urobiome derived bacteriocins could provide many advan­
tages such as enhanced antimicrobial activity, improved yield, and
potentially making bacteriocins more respondent to purification.
Therefore, a mechanistic understanding of microbe-microbe and
microbe-host interactions within the urobiome is still required to gain a
deeper insight into how such potential biological therapeutics truly
interact with the host’s immune system. Ultimately, this would provide
a means of identifying potential biomarkers for non-antibiotic therapies
and combined with bioengineering strategies, lead to the development
of more effective treatments, paving the way of a new era of diagnosis
and therapy in urology.
Funding
Ciara Kenneally is in receipt of a RISAM PhD Scholarship at Munster
Technological University.
12
Microbiological Research 259 (2022) 127010
Conflicts of interest
The authors declare that there are no conflicts of interest regarding
the publication of this paper of which we are aware.
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