c Consult author(s) regarding copyright matters · 2021. 8. 7. · 53 2009, Hola et al., 2012, Stahlhut et al., 2012), and are often associated with enhanced 54 antibiotic resistance

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Carey, Alison, Tan, Chee Keong, Ipe, Deepak, Sullivan, Matthew, Cripps,Allan, Schembri, Mark, & Ulett, Glen(2016)Urinary tract infection of mice to model human disease: Practicalities, im-plications and limitations.Critical Reviews in Microbiology, 42(5), pp. 780-799.

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Abstract 1

Urinary tract infections (UTIs) are among the most common bacterial infections in 2

humans. Murine models of human UTI are vital experimental tools that have helped to 3

elucidate UTI pathogenesis and advance knowledge of potential treatment and infection 4

prevention strategies. Fundamentally, several variables are inherent in different murine 5

models, and understanding the limitations of these variables provides an opportunity to 6

understand how models may be best applied to research aimed at mimicking human 7

disease. In this review, we discuss variables inherent in murine UTI model studies and 8

how these affect model usage, data analysis and data interpretation. We examine 9

recent studies that have elucidated UTI host-pathogen interactions from the perspective 10

of gene expression, and review new studies of biofilm and UTI preventative approaches. 11

We also consider potential standards for variables inherent in murine UTI models and 12

discuss how these might expand the utility of models for mimicking human disease and 13

uncovering new aspects of pathogenesis. 14

15

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Introduction 16

Urinary tract infections (UTIs) are some of the most common bacterial infections in 17

humans and a major burden to health care systems. UTIs cause an estimated 11 million 18

doctor visits in the USA annually, and over 150 million doctor visits globally, with an 19

annual cost upwards of $6 billion (Stamm and Hooton, 1993, Stamm and Norrby, 2001, 20

Foxman, 2003, Bower et al., 2005, Nielubowicz and Mobley, 2010). Categories of 21

disease include complicated and uncomplicated infections, which are further classified 22

according to symptoms and diagnostic markers as urethritis, acute and chronic cystitis, 23

prostatitis, and pyelonephritis (Hay and Fahey, 2002, Kaufmann and Modest, 2002, 24

Neild, 2003, Nicolle, 2008). Complicated UTIs are defined by their association with 25

structural or functional abnormalities of the urinary tract that lead to enhanced 26

susceptibility to infection (Johansen et al., 2011). UTI can progress to life-threatening 27

disease including urosepsis in the absence of appropriate treatment (McBean and 28

Rajamani, 2001, Siegman-Igra et al., 2002). The UTI spectrum also encompasses 29

asymptomatic bacteriuria (ABU); this is a carrier state that resembles commensalism 30

and often leads to the recovery of >105 colony forming units (cfu) per ml of a single 31

bacterial strain from urine without clinical symptoms. 32

33

UTIs occur most frequently in women; over half of all women will experience a UTI in 34

their lifetime (Foxman, 2002, Bower et al., 2005), and up to 30% of these women will 35

experience a recurrent infection that may progress to chronic disease (Stamm and 36

Hooton, 1993, Kunin, 1994, Hooton et al., 1996, Brown, 1999, Stapleton, 1999, Stamm 37

and Norrby, 2001). The incidence of UTI in women over the age of 65 is 21% compared 38

to only 12% in men of similar age (Lee and Neild, 2007). The incidence of specific types 39

of UTI is also disproportionately associated with age and predisposing factors. For 40

example, ABU is overrepresented in several populations including elderly 41

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institutionalized individuals and adult women with diabetes mellitus (Lee and Neild, 42

2007, High et al., 2009, Johansen et al., 2011, van Duin, 2012, Ipe et al., 2013, 43

Schneeberger et al., 2014). A population with a nearly 100% prevalence of ABU is 44

users of chronic indwelling urinary catheters (Warren et al., 1982). The major 45

determinant for development of catheter-associated bacteriuria (CAB) is the duration of 46

catheterization (Nicolle, 2014). Symptomatic catheter associated UTIs (CAUTIs; as 47

advocated by the CDC (Press and Metlay, 2013)) are important among hospitalized 48

individuals, and in these patients, infection with a broad range of pathogens, including 49

mixed infections, is common (Foxman, 1990, Trautner et al., 2005, Jacobsen et al., 50

2008, Meddings et al., 2010, Trautner, 2010). CAUTIs as nosocomial infections have 51

been linked to biofilm formation (Maki and Tambyah, 2001, Ong et al., 2008, Frank et al., 52

2009, Hola et al., 2012, Stahlhut et al., 2012), and are often associated with enhanced 53

antibiotic resistance (Johnson et al., 1999, Wagenlehner and Naber, 2004, Chenoweth 54

and Saint, 2013). 55

56

Causal agents of UTIs 57

Uropathogenic Escherichia coli (UPEC) cause up to 80% of all UTIs (Ronald, 2002, 58

Stamm, 2006), particularly among young women (Salvatore et al., 2011). The major 59

reservoir for UPEC is the gastrointestinal tract, with spread to the genital tract and 60

urethral opening providing an opportunity for bladder colonization (Stamm, 2006). Other 61

noted etiological agents of UTI include Klebsiella spp., Pseudomonas spp., Citrobacter 62

spp., and Proteus spp. (Ronald, 2003, Lee and Neild, 2007, Tabibian et al., 2008). 63

Several Gram-positive bacteria including Staphylococcus spp., Enterococcus sp., and 64

Streptococcus agalactiae (also known as group B streptococcus; GBS) also cause UTI 65

and ABU (Raffi et al., 2005, Singh et al., 2007, Tena et al., 2007, Natarajan, 2008, 66

Alamuri et al., 2009, Ulett et al., 2009, Tan et al., 2012b). Some pathogens are more 67

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frequently associated with distinct UTI clinical conditions and patient groups (Ronald, 68

2002, Ipe et al., 2013). For example, Staphylococcus saprophyticus affects mainly 69

younger women, Morganella morganii, Providencia stuartii, Pseudomonas aeruginosa, 70

Enterobacter and Serratia sp. are frequently associated with severe UTI (Johnson et al., 71

1987, Stamm and Hooton, 1993, Franco, 2005, Nielubowicz and Mobley, 2010), and 72

enterococci are overrepresented among complicated UTI and hospital-acquired CAUTIs 73

(Warren et al., 1982, Barnett and Stephens, 1997, Ronald, 2002, Tabibian et al., 2008). 74

Nosocomial CAUTI, which is often associated with mixed bacterial species, is caused 75

by a more diverse range of Gram-negative and Gram-positive pathogens, including E. 76

coli, Proteus spp., Pseudomonas spp., Providencia stuartii, Staphylococcus spp., and 77

Enterococcus spp. (Sillanpaa et al., 2010, Nielsen et al., 2012). While E. coli causes 78

most ABU, Klebsiella spp., enterococci, streptococci and Candida spp. are also 79

relatively common among certain patient groups (Ronald, 2002, Ronald, 2003, Ipe et al., 80

2013). 81

82

Insights into UTI pathogenesis from studies using mice 83

In order to successfully colonize the urinary tract, pathogens must endure a multitude of 84

host factors that mediate localized defence to prevent UTI (Schilling et al., 2001a, Song 85

and Abraham, 2008, Ragnarsdottir et al., 2011, Ulett et al., 2013). In addition to the 86

normal urinary flushing mechanisms and the (impermeable) urothelial-lining of the 87

bladder, factors including host antimicrobial peptides, uromodulin (also known as 88

Tamm–Horsfall protein) and cytokines contribute to host defence. 89

90

Substantial advances in our understanding of the pathogenesis of UTI have come from 91

the development and characterization of murine models of human infection. Different 92

versions of the murine UTI model have been used to dissect the roles of cytokines in 93

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innate defence including CCL2 (Ingersoll et al., 2008, Sivick et al., 2010), CCL4 94

(Ingersoll et al., 2008), IL-1β (Ingersoll et al., 2008, Ulett et al., 2010), IL-6 (Hedges et 95

al., 1992, Wullt, 2003, Ingersoll et al., 2008, Nielubowicz and Mobley, 2010, Sivick and 96

Mobley, 2010), IL-8 (mouse equivalent CXCL1/2) (Wullt, 2003, Wullt et al., 2003, 97

Ingersoll et al., 2008, Nielubowicz and Mobley, 2010), IL-9 (Kline et al., 2011), IL-10 98

(Duell et al., 2012a), IL-17A (Ingersoll et al., 2008, Sivick and Mobley, 2010), IL-12p40 99

(Ingersoll et al., 2008), G-CSF (Ingersoll et al., 2008, Sivick and Mobley, 2010) and 100

TNF-α (Sivick and Mobley, 2010, Sivick et al., 2010). The synthesis of some of these 101

cytokines begins as early as 1 h post-infection in mice, peaks at 24 h and returns to 102

baseline by 2 weeks. A commonly used model employs the C57BL/6 mouse strain and 103

has illustrated the early cytokine responses that correlate with peak bacterial burdens in 104

the bladder and subsequent UPEC clearance (Sivick and Mobley, 2010). Other models 105

have shown that production of IL-8 (CXCL1/2 in mice) triggers an influx of neutrophils 106

(Hang et al., 2000, Smithson et al., 2005, Artifoni et al., 2007, Sivick and Mobley, 2010, 107

Sivick et al., 2010) via toll-like receptor (TLR)4-dependent and -independent 108

mechanisms (Agace et al., 1993, Hedges et al., 1994, Agace, 1996, Schilling et al., 109

2001a, Schilling et al., 2001b, Wullt et al., 2001, Ragnarsdottir et al., 2008, Sivick and 110

Mobley, 2010). Recently defined elements of acute UTI pathogenesis have been 111

reviewed elsewhere (Sivick and Mobley, 2010, Ulett et al., 2013). Pathogens that can 112

overcome host defences can exploit the bladder and urine as unique growth 113

environments. Indeed, growth in urine has been linked to recurrent cystitis in individuals 114

with voiding defects (Raz et al., 2000, Scholes et al., 2000, Foxman, 2002) and is 115

probably related to microbial traits that provide ‘bacteruric potential’, as discussed 116

elsewhere (Ipe et al., 2013). 117

118

How do responses to UTI in mice correlate with responses in humans? 119

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We can compare key elements of host defence and immune responses to UTI in mice 120

with what is known about human infection to gain insight into the degree to which murine 121

models reflect host-pathogen interactions in patients. TLR-encoding genes (Andersen-122

Nissen et al., 2007, Ragnarsdottir et al., 2010, Yin et al., 2010) and some chemokine 123

receptors (Frendeus et al., 2000, Godaly et al., 2000, Svensson et al., 2011) offer useful 124

paradigms of consistent and divergent responses between mouse and human UTI. The 125

study of TLR4 and CXCR1 in the context of mouse genetics and defence against UTI 126

(Svanborg Eden et al., 1985, Svanborg et al., 2001, Ragnarsdottir et al., 2008, Sivick 127

and Mobley, 2010) in particular, provide validation of murine models for studying human 128

infection. 129

130

TLR4 and the host response to LPS 131

C3H/HeJ mice, which are LPS non-responsive due to mutation in the tlr4 gene and 132

mount poor inflammatory responses compared to C3H/HeN LPS-responsive mice, are 133

highly susceptible to UPEC UTI (Hagberg et al., 1984). Studies using these mice were 134

critical for establishing the signalling pathway from TLR4 that leads to secretion of 135

cytokines such as IL-6 (de Man et al., 1989), chemokines such as macrophage 136

inflammatory protein 2 (CXCL2), and recruitment of neutrophils (Yadav et al., 2010) that 137

help to control UTI. Interestingly, the control of UTI in the bladder depends on TLR4 138

expression on urothelial and stromal cells, and this is needed to drive inflammatory 139

responses even in the presence of hematopoietic cells with intact TLR4 (Schilling et al., 140

2003). TLR4 signalling has also been associated with increased severity of pain due to 141

UPEC UTI in mice (Rudick et al., 2010), whereas less TLR4 activity has been 142

associated with ABU in mice (Fischer et al., 2006) and in humans (Ragnarsdottir et al., 143

2007, Ragnarsdottir et al., 2010), as noted elsewhere (Ulett et al., 2013). Analysis of 144

TLR4 expression in humans with UTI demonstrated that lower TLR4 expression due to 145

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polymorphisms in the promoter attenuates host responses and promotes asymptomatic 146

carriage of UPEC (Ragnarsdottir et al., 2007, Ragnarsdottir et al., 2010, Hernandez et 147

al., 2011). Another study of tlr4 polymorphisms in children associated a TLR4_A896G 148

polymorphism with development of upper UTI and formation of renal scarring in 149

pyelonephritis (Akil et al., 2012). Thus, observations regarding the function of TLR4 in 150

murine UTI largely parallel findings in human infection and indicate that carefully 151

regulated innate immune responses are critical to protect against UTI. 152

153

Host responses involving TLR5 and TLR11 154

TLR5 and TLR11 provide other examples of connect and disconnect respectively, when 155

we compare data derived from murine models and humans. In humans, a polymorphism 156

in the TLR5 gene (TLR5_C1174T) abrogates the flagellin signalling cascade and has 157

been associated with an increased susceptibility to recurrent UTI (rUTI) (Hawn et al., 158

2009). TLR5 is essential to mediate flagellin-induced inflammatory responses in mice 159

with cystitis, which aid in control of infection in both the bladder and kidneys (Andersen-160

Nissen et al., 2007). Thus, similar to TLR4, data regarding the function of TLR5 appear 161

to be comparable between mouse UTI and human infection. In contrast, TLR11 offers 162

useful insight into the limitations of murine models of human UTI. TLR11 is expressed in 163

mice and plays a role in protecting the kidneys against UPEC (Zhang et al., 2004). The 164

exact mechanism by which TLR11 is activated and provides protection in mice is not 165

known. A TLR11 homologue in humans has yet to be identified, which highlights the 166

divergence between human clinical UTI and mice, and the need for caution when 167

extrapolating some findings from mice to humans. 168

169

Cytokine responses in mice and humans 170

Some of the patterns of cytokine production in mice parallel the immune responses that 171

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have been described in human patients with UTI. For example, IL-1β, IL-6, IL-8, IL-10, 172

CCL2, CCL5, and CXC chemokines are produced early during infection in both mice 173

and humans, as illustrated in Fig. 1 (Ko et al., 1993, Candela et al., 1998, Wullt et al., 174

2002, Roelofs et al., 2006, Godaly et al., 2007, Ingersoll et al., 2008, Sivick and Mobley, 175

2010, Ulett et al., 2010, Duell et al., 2012a, Garcia et al., 2013). Likewise, intracellular 176

bacterial communities (IBCs) occur in mice (Anderson et al., 2003, Rosen et al., 2007) 177

and have been observed in tissues/cells collected from humans with UTI (Rosen et al., 178

2007, Robino et al., 2013, Robino et al., 2014). Fimbrial adhesins of UPEC mediate 179

binding to mouse and human urothelial cells, and play a major role in colonization of the 180

urinary tract (Connell et al., 1996, Mulvey, 2002, Nielubowicz and Mobley, 2010). There 181

have also been recent discoveries regarding the involvement of mast cells in murine 182

UTI that align with recent insight into the human response to infection. In mice, mast 183

cells contribute to the suppression of humoral and cell mediated immune responses in 184

the bladder via the production of IL-10, which was shown to contribute to long-term 185

persistence of UPEC (Chan et al., 2013). Biopsies of human bladder collected from 186

patients with rUTI also contained increased mast cells, suggesting that they play a role 187

in chronic inflammation and contribute to re-occurrence of infection (Chuang and Kuo, 188

2013). A summary of the consistent features in UTI pathogenesis between mice and 189

humans is presented in Fig. 1. 190

191

Bacterial manipulation of the host response 192

UPEC can also pro-actively manipulate key host responses during UTI. For example, 193

UPEC production of TcpC, a Toll/interleukin-1 receptor (TIR) homology domain-194

containing protein, results in inhibition of downstream TLR signaling and enhances 195

UPEC survival in mouse macrophages (Cirl et al., 2008). The effect of UPEC survival in 196

macrophages towards the pathogenesis and severity of UTI is unknown. Interestingly, 197

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however, there are differences between the ability of different E. coli strains to survive in 198

human and mouse macrophages; UPEC UTI89 and ABU VR50 display better survival 199

than UPEC CFT073 and ABU 83972 in murine bone marrow-derived macrophages, 200

whereas in human monocyte derived macrophages, the intramacrophage fitness trait of 201

ABU VR50 is absent (Bokil et al., 2011). This suggests that host species differences 202

may impact on UTI pathogenesis in the context of macrophage anti-microbial pathways. 203

204

Some UPEC strains can also suppress urothelial cytokine responses, subduing the 205

secretion of IL-8 (CXCL8) from human urothelial cells, and inhibiting the transcription of 206

the mouse equivalents, keratinocyte cytokine (KC/CXCL1) and macrophage 207

inflammatory protein 2 (CXCL2) in vivo (Billips et al., 2007). UPEC α-hemolysin 208

promotes shedding of urothelial cells in vitro for both murine and human cells, and 209

suppresses the secretion of the inflammatory cytokine, IL-6, from human bladder 210

urothelial cells (Dhakal and Mulvey, 2012, Hilbert et al., 2012). Curli expression in 211

UPEC also alters the innate immune response by interacting with and binding to the 212

human antimicrobial peptide cathelicidin LL-37, and the murine ortholog, cathelicidin-213

related antimicrobial peptide (CRAMP). Binding of antimicrobial peptide to UPEC curli 214

prevents the former from binding to the bacterial membrane and mediating cell lysis 215

(Kai-Larsen et al., 2010). Thus, LL-37/CRAMP are secreted in urine in response to 216

UPEC and provide protection against UTI in human cell lines in vitro and in mice 217

(Chromek et al., 2006). The concentration of LL-37 in urine was suggested to influence 218

susceptibility to UTI (Nielsen et al., 2014). Other virulence mechanisms used by UPEC 219

to subvert host responses are reviewed elsewhere (Hunstad and Justice, 2010, 220

Nielubowicz and Mobley, 2010, Sivick and Mobley, 2010, Hannan et al., 2012, Ulett et 221

al., 2013). 222

223

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Evaluating fundamental variables in murine UTI models 224

Murine models of UTI represent a crucial link in the ability to study host-pathogen 225

interactions and devise and evaluate new preventive and treatment strategies to protect 226

against infection (Mastroeni and Sheppard, 2004, Mylonakis et al., 2007, Silva et al., 227

2011, Hoelzer et al., 2012, Louz et al., 2013). Mice are low cost, easy to handle, and 228

can be used with a wealth of readily available reagents and tools. Genetically modified 229

strains for studying single gene function also provide considerable benefits and are 230

increasingly being used for UTI pathogenesis studies (Saemann et al., 2005, Anderson 231

et al., 2010, Fischer et al., 2010, Sivick et al., 2010, Yadav et al., 2010, Duell et al., 232

2012a). A summary of murine UTI models used to study UPEC pathogenesis, treatment, 233

prevention and catheter colonization is provided in Table 1. This is intended to comprise 234

illustrative examples of the variables in murine UTI models in detail and is not intended 235

to represent an exhaustive list of all the studies that have used murine UTI models to 236

date. 237

238

The first murine model of human UTI was described in 1975 (Kalmanson et al., 1975). 239

Refinements to this original model in the 1980’s (Iwahi et al., 1982, Hagberg et al., 1983, 240

O'Hanley et al., 1985), and more recently (Hung et al., 2009) have helped to define how 241

these models reflect human infection. The fundamental variables inherent in murine UTI 242

models can by grouped into three categories: (i) mode of infection, (ii) inoculum 243

properties (infectious dose, volume, pathogen and strain), and (iii) mouse strain. Below, 244

we discuss these variables with a focus on carefully defining UTI models and provide an 245

overview of recent investigations that have utilized these models to study UTI. 246

247

Variable 1: mode of infection 248

A range of inoculation procedures has been used to establish infection in the mouse 249

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urinary tract. Variable parameters include the mode of injection, anaesthetic conditions, 250

surgical interventions [e.g. implantation of an indwelling catheter (Li et al., 2002)], post-251

inoculation procedures to restrict voiding [e.g. transient obstruction using collodion film 252

(Johnson et al., 1993a, Tratselas et al., 2014), catheter clamping (Tsuji et al., 2003, 253

Bates et al., 2004, Raffi et al., 2009), or water restriction (Tsuji et al., 2003, Bates et al., 254

2004, Chassin et al., 2006, Raffi et al., 2009, You et al., 2009, Li et al., 2010, Vimont et 255

al., 2012)], dietary modifications [e.g. antibiotic supplementation in drinking water 256

(Schilling et al., 2002, Johnson et al., 2006, Hannan et al., 2010)], operator skill, and 257

lastly, general animal handling and housing conditions. 258

259

The most common mode of infection is inoculation by transurethral (intra-bladder, 260

intravesical) delivery of bacteria directly into the bladder following insertion of a small 261

catheter (~0.6 mm diameter) through the urethral orifice of female mice. Here, the 262

catheter is inserted through the urethral orifice, beneath the pelvic bone and through the 263

external and internal urethral sphincters (total distance ~1.2 cm; the female mouse 264

urethra is ~1 cm long, as measured in 8-10 week old mice (Hopkins et al., 1995)) into 265

the bladder until resistance is felt, then withdrawn several mm prior to direct inoculation 266

into the bladder lumen (Johnson and Brown, 1996, Hung et al., 2009). In humans, all 267

urethral catheterization is called simply that: urethral (or bladder, or urinary) and is 268

always is transurethral (i.e., into the bladder). Thus, transurethral is the term that we 269

have used here to define the inoculation method in mice that most closely parallels 270

catheterization in humans. Alternatively, insertion of the catheter can be restricted to 271

only several mm (~0.5 cm) through the urethral orifice and through the external urethral 272

sphincter (but not through the internal urethral sphincter) to achieve intra-urethral 273

delivery in mice (Hopkins et al., 1995, Hung et al., 2009). However, there is no intra-274

urethral variant of catheter insertion in humans (as in mice). Transurethral inoculation 275

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into the bladder by deeper catheterization may promote bacterial binding to urothelial 276

cells better than intra-urethral challenge and therefore may lead to higher bacterial 277

recovery (Hung et al., 2009). However, no studies have directly compared these 278

approaches in side-by-side trials. A third inoculation approach is also possible; 279

perurethral inoculation, which can be achieved superficially at the urethral office. In this 280

approach, the urethra is not cannulated because the catheter is placed only ~0.2 cm 281

within the urethral orifice and does not pass beneath the pelvic bone, nor through the 282

external or internal urethral sphincter (i.e. pelvic diaphragm). There is no perurethral 283

variant of catheter insertion in humans; but this approach may provide a parallel to UTI 284

in humans where infection develops in the absence of transurethral catheterization. The 285

three inoculation methods, illustrating placement of the catheter tip, are shown in Fig. 2. 286

287

The speed of delivery of the inoculum (i.e. challenge force) may also affect bacterial 288

dispersal following inoculation of the bladder (Johnson and Brown, 1996, Russo et al., 289

1996a, Johnson, 1998, Hvidberg et al., 2000, Hung et al., 2009). To manage this, 290

electronically or mechanically regulated injection pumps have been used to control the 291

speed of delivery of the inoculum (Mobley et al., 1993, Johnson and Brown, 1996). The 292

most commonly used method involves the use of a 1-mL syringe directly attached to a 293

catheter for manual injection of inoculum volumes ≤50 µl to prevent vesicoureteral reflux 294

(VUR), as discussed further below. 295

296

Alternative infection procedures that do not use transurethral or intra-urethral delivery 297

have been applied in some studies. For example, surgical opening of the peritoneal 298

cavity was used to facilitate injection of the inoculum into the bladder followed by 299

suturing and recovery in one study (Nicholson and Glynn, 1975). Another mini-surgical 300

bladder inoculation technique yielded reliable infection results for the upper and lower 301

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urinary tract without systemic complications or peritonitis and offers excellent potential 302

for advancing the study of UTI in male mice (Olson and Hunstad, 2014). It is noteworthy 303

however that these methods probably best parallel transabdominal surgery through the 304

suprapubic region. Therefore, it is conceivable that these models could lead to atypical 305

disease manifestations as a reflection of non-physiologic means of (natural) bladder 306

infection whereby bacteria originate from the periurethra and ascend into the bladder. 307

308

Suprapubic catheterization is a common surgical procedure for patients where urethral 309

catheterization is not recommended (Tenke et al., 2014). Complications from this 310

include urine leakage, hematuria and urine retention, which are unlikely to be 311

associated with transurethral catheterization (Healy et al., 2012); more practically, this 312

procedure, independent from transabdominal surgery, has not yet been reported in mice. 313

Future studies that analyse the incidence of complications arising from these different 314

infection procedures in mice would thus be of interest. More importantly, studies on male 315

mice will now be possible and may help to elucidate the basis of recently reported sex 316

differences between males and females for the development of more severe UTI (Olson 317

and Hunstad, 2014). 318

319

Finally, bloodstream infection by intravenous infection in a model of haematogenous 320

spread of streptococci to the urinary tract was used in one study to provide a model of 321

urosepsis (Furtado, 1976). In humans, bloodstream infection arising from a primary UTI 322

represents ascending infection with secondary dissemination; bloodstream challenge 323

murine models represents haematogenous infection and descending UTI. Thus, while 324

streptococci do cause pyelonephritis and urosepsis (Ulett et al., 2009, Ulett et al., 2012), 325

murine models of haematogenous infection and descending UTI are minimally 326

applicable to the human situation since this infection approach does not parallel the 327

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natural progression to urosepsis in patients. 328

329

A final comment on the limitation of the direct mode of infection in murine models is also 330

warranted since, as noted, bacteria typically originate from the periurethra and ascend 331

into the bladder to cause UTI in humans. Thus, the factors that determine vaginal and 332

periurethral colonization in humans, and bacterial entry from the periurethral area into 333

the bladder, are bypassed by direct instillation of bacteria into the urinary tract in murine 334

models. The importance of this disconnect with murine UTI models remains unknown. 335

336

Variable 2: inoculum properties 337

Challenge inoculum is a second key variable in murine UTI models and is summarized 338

according to pathogen and strain, infectious dose and challenge volume in Table 1. 339

Typically, the infectious dose in transurethral models is 107-109 cfu; however, there is 340

considerable variation in this dose and studies have used as few as 103 cfu, and up to 341

1010 cfu (Table 1). An infectious dose of 108 cfu is common, and for studies that enrich 342

UPEC cultures for type 1 fimbriae expression by serial static subculture, a dose of 107 343

cfu is frequently used. This lower dose is linked to the higher expression of type 1 344

fimbriae, and their role in mediating adherence to uroplakins on superficial bladder 345

epithelial cells (Wu et al., 1996, Nielubowicz and Mobley, 2010, Cusumano et al., 2011). 346

The incidence of persistent bacteriuria in C3H/HeN mice that receive ten-fold more type 347

1 fimbriae-enriched UPEC to establish chronic infection (108 vs 107 cfu) is increased, 348

but the higher dose does not alter kidney-bladder titre ratios after one month (Hannan et 349

al., 2010). These data show that infectious dose can influence model read-outs in 350

separate compartments of the urinary tract independently. In interpreting data from 351

different murine models one also needs to consider that the effects of infectious dose 352

can change as a function of host background. For example, the progression to chronic 353

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cystitis is dose-dependent in C3H/HeN mice but not C57BL/6 mice (Hannan et al., 354

2010). Thus, adjustments to the standard inoculation approach of a single infectious 355

dose of 108 cfu (or 107 cfu type 1 fimbriae-enriched cultures) may be useful for different 356

pathogens or specific studies; for example, uropathogenic streptococci were 357

administered at a high infectious dose of 109 cfu to achieve consistent ID90 outcomes 358

(Tan et al., 2012a), and differential dosing was useful to model the effects of specific 359

virulence factors including flagella in a study on UPEC (Lane et al., 2007). A more 360

recent study focused on comparing a single infectious dose with superinfection in 361

C3H/HeN mice and C57BL/6 mice (Schwartz et al., 2015). Remarkably, superinfection 362

of C57BL/6 mice with UPEC UTI89 rendered these mice (that are normally resistant to 363

chronic cystitis following a single infection) significantly more susceptible to developing 364

persistent bacteriuria and chronic cystitis; serum levels of IL-6, KC/CXCL1, and G-CSF 365

predicted the development of chronic cystitis. Notably, however, the response to infection 366

differed between C3H/HeN and C57BL/6J mice, especially for immunoglobulin genes. 367

368

Inoculum volumes typically range between 10-100 µl (Table 1) (Nicholson and Glynn, 369

1975, Furtado, 1976, Hagberg et al., 1983, Hopkins et al., 1995, Hung et al., 2009, Thai 370

et al., 2010). Differences in volume affect early events in bladder colonization. Most 371

notably, the UTI condition being studied (cystitis, pyelonephritis, urosepsis, VUR) should 372

guide the choice of volume. A standard of 25-50 µl is typical and is increased to 100-373

150 µl to model pyelonephritis and induce VUR (Table 1) (Hopkins et al., 1998, Johnson, 374

1998, Morin and Hopkins, 1998). VUR in murine models most likely relates to both the 375

(larger) inoculum volume and the (increased) force of injection/speed that causes reflux 376

to the kidneys (Hopkins et al., 1995); intra-urethral or, alternatively, superficial 377

perurethral challenge (at the urethral orifice), may not induce VUR as readily as forceful 378

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deeper transurethral injection directly into the bladder (Johnson and Brown, 1996, 379

Hopkins et al., 1998, Johnson, 1998, Hung et al., 2009). In addition, the potential for 380

high-pressure VUR to induce kidney trauma (e.g. tearing of tissue, and gross 381

haematuria) as a result of the use of larger volumes and increased force has also been 382

documented in the murine UTI model (Johnson and Manivel, 1991). Hydrostatic injury to 383

the kidney creates a non-physiologic substrate for infection that is unrepresentative of 384

uncomplicated pyelonephritis (Johnson and Brown, 1996). Thus, inoculation-induced 385

VUR is not desirable if the goal is to mimic the natural pathogenesis of ascending 386

pyelonephritis, in which bacteria must ascend the ureter(s) themselves, unaided by 387

artificial bladder overpressure. Notably, studies on UPEC and P. mirabilis have shown 388

true ascending UTI in the murine model based on transurethral inoculation procedures 389

that do not induce VUR of the inoculum (i.e. 25-50 µl volume delivered by slow infusion) 390

(Hagberg et al., 1983, Johnson et al., 1993b, Hvidberg et al., 2000). Routine use of a 391

standard volume of 25-50 µl among non-VUR studies will improve the ability to compare 392

studies where colonization data is derived using equivalent inoculation methods. We 393

suggest normative standards for volume and other variables, as well as alternative 394

methods for application-based modifications in Table 2, which should aid standardization 395

procedures for experimental infection in murine UTI models. 396

397

The inoculum properties relating to bacterial strain are a major variable between studies 398

and in many cases, represents the actual variable under study. In some cases, the 399

bacterial strain is simply a way to achieve infection for investigation of aspects of the 400

infection process other than the characteristics of the strain. However, for studies in 401

which the characteristics of the strain are the main focus, the choice of strain is critical. 402

We have summarized the use of different bacterial strains, chiefly for UPEC, in Table 1. 403

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This lists the commonly used prototype strains including the urosepsis strain, CFT073, 404

that was isolated from the blood of a pyelonephritis patient, and the cystitis strain, UTI89, 405

that was isolated from a patient with recurrent cystitis. UPEC are a highly heterogeneous 406

population and different strains possess different sets of virulence factors that are 407

important at different stages of UTI. CFT073 is a highly toxigenic strain that can cause 408

severe damage to the urothelium and immunopathology (Mobley et al., 1990), but can 409

also invade epithelial cells and form IBCs (Garofalo et al., 2007). UTI89 is a more 410

invasive strain that forms IBCs and survives intracellularly, but also expresses several 411

toxins that cause urothelial damage (Chen et al., 2009). The mosaic nature of the UPEC 412

genome means that there are no unique genetic features that clearly distinguish 413

different pathogenicity lifestyles. 414

415

Table 1 also highlights strains for which limited data from murine models are available, 416

such as the recently emerged and globally disseminated multidrug resistant UPEC 417

clone of serotype O25b:H4, sequence type 131 (E. coli ST131) (Totsika et al., 2011). As 418

discussed, type 1 fimbriae are essential for UPEC colonization of the mouse urinary 419

tract and many studies employ UPEC cultures enriched for type 1 fimbriae expression 420

following static growth. Given the importance of this phase-variable phenotype for 421

bladder colonization, we suggest that the level of type 1 fimbriae expression should be 422

carefully monitored, particularly for experiments comparing wild-type and mutant strains. 423

Methods such as yeast cell agglutination (Schembri et al., 2000), fim switch PCR (Gally 424

et al., 1996), hemagglutination (Hagberg et al., 1981) or western blotting using Fim-425

specific antibodies would be appropriate. 426

427

The use of co-infection models, where various strains of bacteria are mixed in the 428

challenge inoculum, have proved highly beneficial in identifying the role that various 429

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virulence factors play in pathogenesis cascades including bladder colonization, the 430

ascension of infection and nutrient acquisition. Co-infection models can involve either i) 431

the use of different wild-type strains, where one is examining interactions between the 432

strains, or comparative fitness, or host responses; or ii) the use of a parent strain and its 433

mutant or transconjugant derivative in an effort to identify the impact of a specific trait on 434

competitive fitness. Competition studies using UPEC and mutants deficient in various 435

genes required for metabolism, for example, showed that the tricarboxylic acid cycle 436

and gluconeogenesis contributes to virulence in vivo (Alteri et al., 2009b). Similar studies 437

have been performed to characterize the role of flagella in UPEC fitness (Lane et al., 438

2005), and autotransporter proteins in bladder colonization (Allsopp et al., 2010, Allsopp 439

et al., 2012). Mixed infections using UPEC mutants lacking iron receptors have 440

demonstrated that the uptake of either aerobactin or yersiniabactin is more important for 441

colonization of the UPEC strain CFT073 than enterobactin or salmochelin utilization 442

(Garcia et al., 2011). However, a different siderophore dependence profile for 443

colonization of the urinary tract was reported for the ABU strain 83972 (Watts et al., 444

2012b). Co-infection with P. mirabilis and P. stuartii was recently shown to increase the 445

incidence of bacteremia in a urease-dependent manner in a murine model of ascending 446

UTI (Armbruster et al., 2014). Studies have also shown that mixed infection with 447

different uropathogens can significantly alter the outcomes of infection. Co-infection of 448

GBS and UPEC, for example, increases the fitness of UPEC in the bladder during acute 449

infection, perhaps through modulation of innate immune responses. This co-infection 450

combination was also shown to increase the risk of developing latent bacterial 451

reservoirs in mice and pyelonephritis (Kline et al., 2012), as well as acute high-titre 452

cystitis in aged multiparous mice (Kline et al., 2014). 453

454

Variable 3: mouse strain 455

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Different mouse strains respond to UTI distinctly and results generated in one strain are 456

not always applicable to other strains. For example, different immune responses in 457

different mouse strains can result in the development of acute or chronic UTI. Divergent 458

responses to UTI in mice were first noted in a comparison of ten inbred strains (Hopkins 459

et al., 1998). The relative susceptibility and resistance phenotypes of commonly used 460

mouse strains were more recently quantitated (Hannan et al., 2010). These data 461

illustrate the major influence of host genetic background, as reviewed elsewhere 462

(Svanborg Eden et al., 1985, Ragnarsdottir et al., 2011). Importantly, dissimilar 463

responses between different mouse strains reflect the genetic diversity in human 464

populations and provides the opportunity to infer clinical relevance based on genetic 465

differences (Hopkins et al., 1998, Ragnarsdottir et al., 2011). In other words, no single 466

mouse strain is ideally suited to mimic all of the diverse requirements needed to model 467

different types of human UTI. A summary of infection and inflammation differences (in 468

terms of severity) between different mouse strains is shown in Fig. 3. 469

470

Most studies have used one of three inbred mouse strains: C57BL/6, CBA/J, or 471

C3H/HeN; often with genetically distinct derivatives or related strains with contrasting 472

susceptibility phenotypes (e.g. C3H/HeJ) (Svanborg Eden et al., 1985). C57BL/6 mice 473

are intermediately susceptible to acute UTI and progressively resolve bacteriuria over 474

14 days, with sporadic and low-level kidney infection (Hopkins et al., 1998, Hannan et 475

al., 2010, Duell et al., 2012a); these mice are resistant to chronic cystitis but exhibit low-476

level bladder colonization (104 cfu/ml) at 14 days after a single infection (Hannan et al., 477

2010). Superinfection can lead to increased rates of persistent bacteriuria and chronic 478

cystitis in the C57BL/6 strain (Schwartz et al., 2015). In addition, C57BL/6 mice provide 479

the advantage of having many gene-deficient mutant derivatives available. CBA/J mice 480

are useful to model ascending UTI and, like DBA/2J and C3H/HeN mice (Schwartz et al., 481

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2015), are susceptible to chronic cystitis and inflammation (Johnson et al., 1998, Snyder 482

et al., 2004, Hagan and Mobley, 2009, Sabri et al., 2009). However, CBA/J mice 483

develop pyelonephritis at a lower frequency compared to C3H/HeN and C3H/HeN-484

derived strains (Hannan et al., 2010). C3H/HeN mice are more susceptible to persistent 485

bacteriuria compared to C57BL/6 mice, and exhibit two, mutually exclusive, outcomes to 486

acute infection: either persistent, high-level bacteriuria (>104 cfu), bladder colonization 487

(>104 cfu) and chronicity over 4 weeks (Hannan et al., 2010); or resolution of bacteriuria 488

(Schwartz et al., 2015). C3H/HeN mice have been particularly useful in studies with the 489

LPS-nonresponsive C3H/HeJ strain to elucidate the role of TLR4 in UTI pathogenesis, 490

as discussed in the section above. 491

492

Approaches to modify innate susceptibility of mice to model specific forms of human UTI 493

have also been developed. For example, the use of chemical destruction of pancreatic 494

islet cells has been used to study diabetes and UTI. C3H/HeN, C3H/HeJ and C57BL/6 495

mice that are rendered diabetic by intraperitoneal injection of streptozocin exhibit 496

increased susceptibility to UTI following infection with UPEC, K. pneumoniae and E. 497

faecalis (Rosen et al., 2008). These data parallel the association between diabetes and 498

increased susceptibility to UTI in adults (Hoepelman et al., 2003). UTI in pregnancy has 499

also been modeled using several strains of mice. In a unique murine model of UTI-500

induced pre-term labor in pregnant C3H/HeJ mice, the survival rates of mothers and 501

pups were influenced by UPEC bearing the Dr adhesin (Kaul et al., 1999). Another 502

study in pregnant C57BL/6J mice showed that despite the inability to culture UPEC from 503

uterine tissue, a pattern of inflammation within uterine tissue was observed and this 504

correlated with a decrease in fetal weight (Bolton et al., 2012). These data suggest that 505

inflammation due to UTI during pregnancy is not localized to the bladder, and may affect 506

urogenital tract tissue more generally. 507

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508

The source of mice may also be important. In one study using outbred Swiss-Webster 509

mice, for example, the investigators reported a supplier-dependent variation in renal 510

infection, as well as a divergence in the development of pyelonephritis (Russo et al., 511

1996a). It is possible that this relates to outbred strains and hybrid vigour (i.e. genetic 512

differences between parents that produce synergistic, favourable effects in offspring), 513

but these findings emphasize the benefits of single-source suppliers and highlight the 514

potential problems that may arise from sub-standard animal care, housing, and quality 515

control. Finally, the influence of distinct anatomy, diet, urinary composition, behaviour, 516

and micturition habits between mice and humans in the context of UTI, and the potential 517

correlation between these factors and pathogen (Nesta et al., 2012), is unclear. An 518

entirely unexplored area is the role of the gut microbiome in influencing susceptibility to 519

UTI. Recent data related to other mucosal sites (Ichinohe et al., 2011, Pang and Iwasaki, 520

2012, Oh et al., 2014) suggest this will be an exciting area for future murine UTI model 521

research. 522

523

Duration of observation post-inoculation is also an important variable related to mouse 524

strain (because of differential susceptibility) (Hannan et al., 2010). There is some 525

evidence of a time-dependent sequence of events after bacterial challenge; observation 526

at a given time point will catch only part of that and the part observed will be determined 527

by the time point chosen. Inoculum properties also relate to the duration of observation 528

post-inoculation since differences in doses and virulence may effect the time course of 529

events after challenge. Particular time points have highlighted the dynamic nature of 530

cytokine production, for example, where some responses peak while other subside at a 531

given time point. Ingersoll et al. (2008) addressed the issue by performing time course 532

studies of bacterial clearance and defining how this relates to host responses (Ingersoll 533

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et al., 2008). For data on human UTI, definitive time course analysis is rarely possible, 534

except in inoculation studies (Wullt et al., 1998, Hull et al., 2000, Sunden et al., 2010, 535

Koves et al., 2014). Lane et al. (2007) used an IVIS to monitor infection in mice in real-536

time, which provides a valuable approach for extended time course studies (Lane et al., 537

2007). Thus, the murine model provides critical information on the dynamics of infection 538

and host responses, which is limited by the duration of observation post-inoculation. 539

540

Transcriptomic studies of UPEC and host responses 541

Analysis of bacterial and host genes that are activated or inactivated during UTI has led 542

to the identification of several key elements of pathogenesis. Similar patterns of UPEC 543

gene expression between in vivo- and in vitro-grown bacteria have been defined. 544

Analysis of UPEC grown in human urine or synthetic media in vitro with UPEC 545

recovered ex vivo from mouse urine or human urine during UTI has also revealed niche-546

dependent gene expression. For example, UPEC grown in human urine in vitro up-547

regulates genes for iron acquisition (e.g. sit, fhuA, chuA), capsular polysaccharide and 548

LPS synthesis (Snyder et al., 2004, Hagan et al., 2010), which parallels UPEC gene 549

expression patterns in mice ( i.e. similar patterns); however, the expression patterns for 550

these genes differ in UPEC grown in LB (i.e. divergent patterns) (Snyder et al., 2004). 551

552

Other niche-dependent patterns of gene expression relate to UPEC genes that encode 553

fimbriae. Enhanced expression of type 1 fimbriae and down-regulation of P fimbriae and 554

flagella expression occurs in mice, but not in human urine cultures in vitro. In the latter, 555

other adhesin and UPEC motility genes including papA_2 (major P fimbriae subunit) 556

and fliC are up-regulated (Snyder et al., 2004). In making such comparisons it is 557

important to consider the experimental conditions in which gene expression is 558

compared, since, for example, the mode of in vitro bacterial growth (e.g. shaking verses 559

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static serial subculture) influences the expression of genes including type 1 fimbriae. 560

Comparisons of gene expression in UPEC isolated from urine of infected mice, UPEC 561

isolated from cystitis patients, and UPEC grown in human urine ex vivo revealed similar 562

gene expression profiles (Hagan et al., 2010). Comparing patient-recovered UPEC 563

strains with those grown ex vivo revealed up-regulation of cell division factors (ftsW), 564

protein secretion components (secD, yidC), and nucleotide synthesis (guaA, pyrG) in 565

vivo, implying faster replication of UPEC during UTI in humans. UPEC isolated from 566

patients also express genes for iron acquisition (chuA, fhuA) and respiration (cyoABCD, 567

cydAB), as occurs in mice. Up-regulation of UPEC genes associated with iron 568

acquisition in IBCs in murine bladder was also reported (Reigstad et al., 2007). Iron 569

acquisition is fundamental to microbe survival in the urinary tract, and the increased 570

transcription of iron acquisition genes by UPEC during IBC formation contributes to 571

UPEC intracellular survival. Conserved UPEC transcriptional responses (Fig. 1) provide 572

a context for future studies that depend on inter-species convergence of bacterial gene 573

expression patterns. 574

575

Divergent gene expression between UPEC in humans and mice can also provide useful 576

insight into potential disease mechanisms by revealing inter-species differences. For 577

example, it was found that genes encoding type 1 fimbriae (fimA, fimH) are highly 578

expressed in UPEC recovered from urine of infected mice but not in UPEC voided in 579

human urine (Snyder et al., 2004, Hagan et al., 2010). However, UPEC do express fimA 580

when grown in human urine in vitro (Hagan et al., 2010). Thus, type 1 fimbriae are 581

required for (and expressed during) infection in mice but their expression during distinct 582

stages of human UTI requires more study. It is possible that adhesin-expressing 583

bacteria may be selectively retained within the urinary tract and not shed in urine, which 584

could complicate conclusions of adhesin non-expression in vivo during UTI. Together, 585

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these studies show that conserved patterns of gene expression in UPEC occur in mice 586

and human UTI ex vivo (recovered) and in vitro (urine assay), as do divergent UPEC 587

responses between mice and humans. Recent studies that profile UPEC gene 588

transcription during human UTI using RNA-seq have advanced our understanding of 589

UPEC pathogenesis (Bielecki et al., 2014, Subashchandrabose et al., 2014). Future 590

studies to functionally define the roles of conserved and divergent UPEC gene 591

expression patterns, including at different stages of infection, will now be essential. 592

593

Recent studies have also probed gene expression in uropathogens other than UPEC 594

using in vitro and ex vivo models. In one study, microarray analysis of Proteus mirabilis 595

isolated from mice demonstrated that genes encoding mannose-resistant Proteus-like 596

fimbriae (mrpA) were upregulated along with genes for amino acid and carbohydrate 597

transporters (dppA, oppA), energy production and iron transport (exbBD, sitABC) 598

(Pearson et al., 2011). Some bacteria can grow efficiently in urine and, as a fitness trait, 599

this can influence the course of UTI, including ABU (Ipe et al., 2013). The expression of 600

several genes in E. coli involved in metabolism has been linked to urine growth (Russo 601

et al., 1996b, Roos and Klemm, 2006, Roos et al., 2006, Alteri and Mobley, 2007, Watts 602

et al., 2012b). Examination of E. faecalis in human urine also demonstrated up-603

regulation of iron transporter genes (feoA, feoB) in this organism (Vebo et al., 2010). 604

Together, these data alongside those for UPEC confirm the importance of genes 605

involved in iron transport and metabolism for fitness in the nutrient poor environment of 606

urine. 607

608

Recent transcriptomic studies in mice have also broadened our knowledge of the host 609

immune response to UTI and revealed responses that are relevant to co-infection. Initial 610

bladder responses are complex with 1564-genes and 2507-genes altered in the bladder 611

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in response to UPEC at 2 h and 24 h, respectively (Duell et al., 2012a, Tan et al., 612

2012a). Strong activation of IL-10 has implications for immune responses during UTI 613

because IL-10 is a master regulator of immunity (Duell et al., 2012b). Direct comparison 614

of gene expression activated by GBS revealed only 172 genes in the response to 615

infection; 68 were also triggered by UPEC. Thus, unique bladder responses are 616

triggered by different uropathogens. Notably, there are few studies on human bladder 617

responses to UTI and responses between mice and humans can differ (Seok et al., 618

2013). A recent study of inflammasomes in the murine bladder urothelium (Hughes et 619

al., 2014), and their role in inflammation and physiology (i.e. urodynamics), highlights an 620

area for future research. 621

622

Studies on virulence factors that impact pathogen-specific immune responses are now 623

needed. For example, UPEC cytotoxic necrotizing factor (CNF1) and hemolysin (HlyA1) 624

induce the expression of several inflammatory mediators in the bladder or urine in mice 625

24 h after infection; including IL-6, CXCL1, CXCL2 and TNF-α (Garcia et al., 2013), 626

which is consistent with the transcriptomic studies discussed above. UPEC infection of 627

the mouse bladder also leads to the up-regulation of genes associated with suppression 628

of transforming growth factor-β and the glycoprotein Wnt5a; both genes are associated 629

with renewal of the bladder epithelium (Mysorekar et al., 2002). This suppression slows 630

regeneration of the uroepithelium and may enhance susceptibility to recurrent infection. 631

It would be of interest to determine whether UPEC infection of the human bladder 632

triggers equivalent gene expression responses. Collectively, these studies have 633

provided important information with regard to host-pathogen interactions and bacterial 634

subversion of the immune response to UTI. Future work employing next-generation 635

sequencing methods (e.g. RNA-seq) will help to further profile the genetic signatures 636

that define transcriptomic changes during UTI. 637

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638

Catheter associated UTI (CAUTI) and Biofilms 639

The use of indwelling catheters results in enhanced predisposition to UTI through the 640

introduction of organisms into the bladder (from the urethra), direct damage to the 641

urothelium, and the provision of an abiotic surface for biofilm formation. Biofilms are 642

recalcitrant to antibiotics and immune responses, a property mediated by multiple 643

factors including the production of a polymeric matrix that is difficult to penetrate. 644

Numerous bacterial factors contribute to the establishment of UPEC biofilms on inert 645

surfaces, including type 1 and some other chaperone-usher fimbriae, and the 646

autotransporter proteins Ag43, UpaG and UpaH (Ulett et al., 2007, Ong et al., 2008, 647

Allsopp et al., 2010, Stahlhut et al., 2012, Murphy et al., 2013). The production of curli 648

and cellulose have also been shown to mediate the formation of biofilms through 649

participation in adherence to human bladder and kidney epithelial cells in vitro and in 650

the early stages of murine UTI (Kai-Larsen et al., 2010, Hadjifrangiskou et al., 2012). 651

652

Recently, a murine model of enterococcal CAUTI was used in which silicone implants 653

were inserted transurethrally, followed by infection (Guiton et al., 2010, Nielsen et al., 654

2012). This model has permitted the identification of new cell-surface factors that 655

contribute to biofilm formation, including the enterococcal surface protein, Esp (Shankar 656

et al., 2001), the pilus-associated sortase A and C (Kemp et al., 2007, Guiton et al., 657

2010) and the endocarditis and biofilm-associated pilus, Ebp (Nielsen et al., 2012). In 658

addition, this method demonstrated that foreign bodies elicit major histological changes 659

in the bladder and local pro-inflammatory cytokine responses. In studies on the 660

application of the model to E. faecalis-mediated UTIs, the investigators demonstrated 661

that the bacteria induce the expression IL-1β and macrophage inflammatory protein 1α 662

(CCL3). These findings represent a significant advance in our understanding of CAUTI 663

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in the host, and underscore the importance of urinary catheterization during E. faecalis 664

uropathogenesis in terms of driving specific pro-inflammatory cytokine responses 665

(Guiton et al., 2010). 666

667

Catheter implantation into the bladder permits the study of bacterial virulence factors 668

that are involved in the formation of biofilms in vivo. A recent study demonstrated that 669

catheter implantation induces the release of the extracellular matrix protein fibrinogen, 670

which can coat catheter surfaces and act as a receptor for E. faecalis Ebp-mediated 671

biofilm formation (Flores-Mireles et al., 2014). The insertion of a catheter also triggers 672

sloughing of the urothelium, which can elicit the re-activation of quiescent intracellular 673

bacteria (Guiton et al., 2012). Urinary catheter implantation in mice that have resolved 674

bacteriuria can lead to recurrent bacteriuria with the original infecting strain. This is 675

believed to be associated with the existence of quiescent intracellular reservoirs of 676

bacteria that provide a source for recurrent infection (Guiton et al., 2012). This model 677

could be useful for the evaluation of new antimicrobial targets for the treatment of 678

CAUTI (Guiton et al., 2010). Finally, the potential effects of analgesia should be 679

considered in models that use chronic administration of analgesics since this has been 680

shown to influence the development of experimental UTI in rats (Vivaldi, 1968). 681

682

Intracellular Bacterial Communities 683

UPEC form IBCs within superficial bladder epithelial cells of mice during the early 684

stages of acute UTI (Mulvey et al., 2001, Anderson et al., 2003). IBCs share several 685

characteristics with biofilms; for example UPEC utilize type 1 pili and Ag43 for the 686

formation of IBCs and produce a matrix comprised primarily of polysaccharide material. 687

Bacteria within an IBC also undergo characteristic morphological changes as seen in 688

biofilm communities on abiotic surfaces (Anderson et al., 2010). IBCs have been 689

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identified in cells obtained from the urine of children with recurrent E. coli UTIs; staining 690

of cells isolated from the urine revealed long filamentous bacteria and disrupted cells 691

with bacteria within and protruding from the cell membrane (Robino et al., 2013, Robino 692

et al., 2014). Similar to this, cells isolated from women with acute UTI or ABU 693

demonstrated the presence of IBCs and characteristic filamentous bacteria (Rosen et 694

al., 2007). Data from murine models suggest that IBCs possess similar properties to 695

biofilms; they protect UPEC from host immune responses and antibiotic treatment, and 696

provide a source for recurrent infection. The use of murine models to examine this 697

aspect of UTI has been invaluable and has provided novel insight into UPEC 698

pathogenesis. 699

700

Treatment and prevention studies in mice 701

Blango and Mulvey (2010) tested seventeen different antibiotics for effectiveness in 702

killing UPEC UTI89 both in vitro and in vivo in relation to IBCs. This study showed that 703

most antibiotics were able to limit UTI89 growth in vitro. However, in their murine UTI 704

model, the authors reported a significantly higher minimum inhibitory concentration for 705

biofilm cultures compared to those observed in vitro, which were associated with IBC 706

formation in the bladder. This led to a conclusion that UPEC persistence in the bladder 707

is related to the entry of UPEC into a quiescent or semi-quiescent state within host cells, 708

and the resilient permeability barrier function associated with the bladder urothelium 709

(Blango and Mulvey, 2010). Other murine UTI models have also been used to examine 710

the efficacy of various treatments. For example, Hvidberg et al. (2000) used eleven 711

clinical isolates of UPEC and one non-pathogenic laboratory E. coli strain to test the 712

therapeutic efficacy of cefuroxime and gentamicin and measured the concentrations of 713

the antibiotics in serum, urine, kidney, and bladder tissues. These studies concluded 714

that while the levels of antibiotics were high in urine, this had little effect on the number 715

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of infecting bacteria in the bladder (Hvidberg et al., 2000). Ultimately, the limitations and 716

advantages of murine models for evaluating new therapeutics in such studies depends 717

on the degree to which these models can mimic human pharmacology and UTI features. 718

719

With the increasing rates of antibiotic resistance, there is an emerging need to identify 720

alternative approaches to prevent UPEC infection of the urinary tract. The use of 721

mannosides, which function as anti-adhesive compounds by preventing FimH-mediated 722

binding to the bladder epithelium, has proven to be effective in the prevention of acute 723

UTI and the treatment of chronic UTI in murine models (Han et al., 2010, Cusumano et 724

al., 2011, Guiton et al., 2012, Totsika et al., 2013). The treatment of mice with 725

mannosides reduces IBC formation in animals with catheter implants and potentiates 726

the effect of antibiotics (Guiton et al., 2012). This approach may provide an alternative 727

to antibiotic therapy that is often ineffective in humans. Importantly, the prophylactic use 728

of the lead mannoside derivative ZFH-04269 prevented the establishment of acute UTI 729

by the multidrug resistant E. coli ST131 strain EC958, and significantly reduced chronic 730

bladder infection in mice following one round of treatment (Totsika et al., 2013). These 731

studies provide strong evidence for the utilization of mannosides in the treatment of UTI 732

caused by multidrug resistant UPEC strains. In a more recent study reported by Hannan 733

et al. (2014), the authors showed that non-steroidal anti-inflammatory drugs such as 734

cyclooxygenase-2 inhibitors may represent an effective treatment and/or preventive 735

strategy as an alterative to antibiotics in certain patients who are susceptible to rUTI 736

(Hannan et al., 2014). Another conceptually new class of therapeutic molecules that 737

target capsule biogenesis was recently used to successfully treat systemic UPEC 738

infection in mice (Goller et al., 2014). 739

740

Murine models have also been used for testing the efficacy of UPEC vaccine antigens. 741

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Multiple animal studies have shown immunogenicity and the presence of vaccine-742

specific antibodies after immunization for several different vaccine approaches, as 743

reviewed elsewhere (Brumbaugh and Mobley, 2012, Moriel and Schembri, 2013). 744

Systemic immunization with FimH afforded long lasting protection against UPEC UTI in 745

one study, leading to a 99% reduction in mouse bladder colonization and kidney 746

infection and high IgG titres in serum and urine (Langermann et al., 1997); similarly 747

FimH fused to FliC, a major flagellin subunit, afforded protection in another murine 748

model (Asadi Karam et al., 2013). Pyelonephritis was prevented by subcutaneous 749

immunization with synthetically produced peptide fragments of digalactoside (Gal-Gal)-750

binding adhesins (pili) of UPEC (Schmidt et al., 1988). In another study, recombinant 751

Gal-Gal pili from four UPEC strains were delivered subcutaneously and afforded 752

protection against homologous and heterologous piliated UPEC strains (Pecha et al., 753

1989). 754

755

Subcutaneous immunization with IroN, a siderophore receptor on E. coli, prevented 756

pyelonephritis but did not provide protection against cystitis (Russo et al., 2003). A 757

large-scale screening and selection process paired with in vivo experiments identified 758

three conserved vaccine candidates, Hma, IutA and IreA (Alteri et al., 2009a). These 759

surface-expressed proteins, all involved in iron acquisition by UPEC, provided 760

protection against challenge with the UPEC strain CFT073. Intranasal immunization 761

with IutA and IreA stimulated antigen-specific IgA secretion into the urine, which was 762

directly correlated to protection in the bladder. Despite antigen-specific IgA responses in 763

the urinary tract of animals immunized with Hma, only the kidneys were protected from 764

infection (Alteri et al., 2009a). Subsequent to this, FyuA, another UPEC iron receptor, 765

was identified as a potential vaccine candidate, protecting from kidney infection through 766

the production of IgG (Brumbaugh et al., 2013). A combination of the four UPEC 767

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surface-expressed iron receptors may prove efficacious as a multivalent vaccine 768

(Wieser et al., 2010). Overall, however the efficacy of experimental vaccines to prevent 769

rUTI in humans has been difficult to establish in the absence of well-structured clinical 770

trials. Still, there is promise; for example, clinical trials of Uro-Vaxom that have been 771

underway for 30-years have demonstrated efficacy for reducing the incidence of rUTI 772

(Brumbaugh and Mobley, 2012). Data derived from murine UTI models (Bosch et al., 773

1988, Baier et al., 1997, Huber et al., 2000, Bessler et al., 2009, Bessler et al., 2010) 774

provide the opportunity for complementary insight into such clinical trials. Finally, a 775

recent study reported successful blocking of the binding of E. faecalis to fibrinogen to 776

prevent catheter-associated bladder infection in mice as a novel vaccine approach for 777

this type of UTI (Flores-Mireles et al., 2014). 778

779

Are human-mouse chimeras the model of the future? 780

Finally, future studies aimed at the development of UTI models utilizing human-mouse 781

chimeras could serve as a valuable tool to provide mouse models that more closely 782

parallel human UTI. Severe combined immunodeficient (SCID)-hu mice have been used 783

to model host-pathogen interactions in various forms (Davis and Stanley, 2003). In one 784

study, SCID mice were more susceptible to UTI than nude mice (Hopkins et al., 1993). 785

While this indicates a lack of thymus-derived T cells does not predispose to UTI 786

(Hopkins et al., 1993, Jones-Carson et al., 1999) the differences in lymphocyte 787

responses between mice and humans (Deknuydt et al., 2009, Ness-Schwickerath and 788

Morita, 2011) imply a need to study SCID mice further in UTI chimera models to explore 789

these findings. There is also the possibility of utilizing xenograft models in the future if 790

human tissues or cell samples can be acquired. An intestinal model using SCID mice 791

has been successfully developed where human intestinal xenografts were placed into 792

the subcapsular regions of the mice and allowed to develop (Savidge et al., 1995). This 793

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was used to examine responses to various infections, including Shigella (Zhang et al., 794

2001). In theory, such an approach could be adapted for the urinary tract using similar 795

methods; the limiting factor being the availability of human bladder tissue. Murine 796

models of this type could help to verify observations from other standard murine models 797

of UTI. 798

799

Conclusions 800

Murine UTI models represent a powerful method to study human UTI. It is vital to 801

consider the variables encompassed in these models and the manner in which they 802

relate to human UTI. In this review, we provide a framework that should aid better 803

standardization of procedures to study UTI. In particular, normative standards for the 804

variables discussed herein including dose, volume, inoculum, injection and 805

helpprocedures post-challenge should be applied where possible to aid standardization 806

of methods in studies. This includes application-based modifications to the standard 807

method (i.e. single 108 cfu dose in 25-50 µl PBS, using deep transurethral insertion and 808

manual injection with no restrictions on inoculum voiding) in the context of alternative 809

strategies, such as those aimed at modeling VUR, super-infection and urosepsis. Some 810

alternatives to this standard method may include a lower infectious dose, repeat dose(s), 811

a higher inoculum volume (e.g. to model pyelonephritis), the use of an indwelling 812

catheter, alternate challenge method (e.g. i.p. delivery to model urosepsis), or 813

restrictions on inoculum voiding post-challenge. Consideration of these methods and 814

alternatives will help to bring the central variables in murine UTI models to the forefront 815

of studies toso that we may improve data interpretation and study design. All of the 816

murine models described in this review will continue to advance the UTI field by 817

providing knowledgeoffering data to inform and guide new pathogenesis, treatment and 818

preventative studies for this extremelythese very common human infection. 819

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infections. 820

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Declaration of Interest: 821

This study was supported with funding from the National Health and Medical Research 822

Council of Australia (APP1084889). AJC is supported by a National Health and Medical 823

Research Council of Australia Peter Doherty Australian Biomedical Fellowship 824

(APP1052464); CKT is a Prime Minister’s Endeavour Asia Fellow; MAS and GCU are 825

supported by Future Fellowships from the Australian Research Council (FT100100662 826

and FT110101048, respectively). There are no other declarations of interest. 827

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Figure 1: Similarities between murine models of UPEC UTI and human infection.

Similarities in the host response to UPEC UTI between mouse and human include

involvement of some UPEC virulence factors and gene expression, host cellular

responses, and cytokine/chemokine signalling. There are also a few UPEC virulence

genes that are expressed differently between mouse and human, such as fimA and

fimH that encode for production of type 1 fimbriae (see text page 17). References

are listed in the right column as: 1: (Anderson et al., 2003); 2: (Rosen et al., 2007); 3:

(Robino et al., 2013); 4: (Snyder et al., 2004); 5: (Hagan et al., 2010); 6: (Mulvey,

2002); 7: (Chan et al., 2013); 8: (Chuang and Kuo, 2013); 9: (Kai-Larsen et al., 2010);

10: (Cusumano et al., 2011); 11: (Agace, 1996); 12: (Wu et al., 1996); 13: (Hang et

al., 2000); 14: (Smithson et al., 2005); 15: (Nielubowicz and Mobley, 2010); 16:

(Schilling et al., 2001b); 17: (Ko et al., 1993); 18: (Candela et al., 1998); 19: (Wullt et

al., 2002); 20: (Roelofs et al., 2006); 21: (Godaly et al., 2007); 22: (Ingersoll et al.,

2008); 23: (Sivick and Mobley, 2010); 24: (Ulett et al., 2010); 25: (Duell et al., 2012a);

26: (Garcia et al., 2013); 27: (Billips et al., 2007); 28: (Ragnarsdottir et al., 2008); 29:

(Duell et al., 2013); 30: (Tan et al., 2012a). Abbreviations are: IL: Interleukin; PMN:

polymorphonuclear cells; CRAMP; murine cathelin-related antimicrobial peptide; IBC:

intracellular bacterial community; TLR: toll-like receptor.

Figure 2: Three different inoculation techniques for murine UTI. The procedure

can be performed using transurethral (A), intra-urethral (B) or perurethral (C)

inoculation methods. The diagram illustrates placements of the tip of the catheter for

A-C in the context of the urethral orifice, urethral sphincters, urethra and bladder.

Figure 3: The severity of inflammation and time course of UPEC bladder

infections in different mouse strains. The time course of infection was derived

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from comparison of mice transurethrally infected with 108 CFU UPEC (Hopkins et al.,

1998, Hannan et al., 2010). The incidence of persistent bacteriuria in the mouse

strains, presented in the study by Hannan et al. (2010), was used to numerically rank

the strains from lowest to highest incidence. This numerical ranking was also done

for the levels of bladder infections in the same strains of mice (Hannan et al., 2010).

The average of these two rankings was calculated and used to generate an ordered

list of mouse strains that resolve infection, or are more likely to develop persistent

infection. This list was compiled with the results of the levels of bladder infections

reported by Hopkins et al. (1998) at the day 14 time point. Similarly, the severity of

inflammation based on inflammatory scores reported by Hopkins et al. (1998) at day

14 post infection, and the mouse strains that were examined for inflammation by

Hannan et al. (2010) were ranked and compiled. While some of the mouse strains

ranked differently between the two studies, the results from Hannan et al. (2010)

were considered to be actual measures of persistence and were used preferentially.

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Table 1. Summary of variables in recent and representative murine UTI models used for the experimental modelling of human UTI

Bacteria Strain Dose (cfu) Volume (µL) Mouse strain Reference

- Pathogenesis and Host Response Studies -

UPEC CFT073 5 × 107 50 C57BL/6 (Sivick et al., 2010)

UPEC CFT073 5 × 107 50 C57BL/6 (Andersen-Nissen et al., 2007)

UPEC CFT073 108 100 C57BL/6 (Yadav et al., 2010)

UPEC CFT073 5 × 108 20 C57BL/6 (Allsopp et al., 2010)

UPEC CFT073 5 × 108 20 C57BL/6 (Allsopp et al., 2012)

UPEC CFT073 5 × 108 25 C57BL/6 (Ulett et al., 2007)

UPEC CFT073 5 × 108 25 C57BL/6 (Tree et al., 2008)

UPEC CFT073 5 × 108 20 C57BL/6 (Totsika et al., 2009)

UPEC CFT073 108 100 C57BL/6, 129/SvJ (Chromek et al., 2006)

UPEC CFT073 109 100 C57BL/6 (Fischer et al., 2010)

UPEC CFT073 3 × 109 40 C57BL/6, CBA (Duell et al., 2012a)†

UPEC, ABU E. coli CFT073, 83972 108 100 C57BL/6J (Jaillon et al., 2014)

UPEC UTI89 107 50 C57BL/6 (Ingersoll et al., 2008)

UPEC UTI89 107 50 C57BL/6 (Danka and Hunstad, 2014)

UPEC UTI89 107 50 C57BL/6 (Wang et al., 2013)

UPEC UTI89 107 50 C57BL/6 (Wang et al., 2014)

UPEC J96 108 50 C57BL/6 (Springall et al., 2001)

UPEC 1677 109 100 C57BL/6 (Roelofs et al., 2006)

UPEC EC958 5 × 108 50 C57BL/6 (Totsika et al., 2011)

UPEC 1177 5 × 107 100 BALB/c (Godaly et al., 2000)

UPEC 1177 108 100 BALB/c (Svensson et al., 2011)

UPEC 1177 1-2 × 108 100 BALB/c (Hang et al., 2000)

UPEC 1177 107 100 BALB/c (Frendeus et al., 2000)

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UPEC 1677 108 50 BALB/c, C3H/HeJ, C57BL/6, DBA.1, DBA.2, C3H/OuJ, SJL, SWR, C3H/HeN, AKR

(Hopkins et al., 1998)

UPEC Clinical 109 50 CBA/J (Johnson et al., 1998)

UPEC, E. coli CFT073, K12 109 25 CBA/J (Sabri et al., 2009)

UPEC, E. coli CFT073, clinical strains, MG1655

108 50 CBA/J (Hagan et al., 2010)†

UPEC CFT073 107 or 109 50 CBA/J (Lane et al., 2007)

UPEC CFT073 2 x 109 20 CBA/J (Barbieri et al., 2014)

UPEC CFT073 5 × 109 50 CBA/J (Snyder et al., 2004)†

UPEC CFT073 108 50 CBA/J (Hagan and Mobley, 2009)

UPEC C1, C2b 5 x 108 25 CBA (Sidjabat et al., 2009)

UPEC, ABU E. coli CFT073,1177, 536, NU14, 83972

109 25 CBA (Roos et al., 2006)

UPEC UTI89 108 50 C3H/HeN, C3H/HeJ (Schilling et al., 2001b)

UPEC UTI89 5 × 106-2 × 107 50 C3H/HeN, C57BL/6J (Schwartz et al., 2015)†

UPEC, GBS UTI89, COH1 1-2 × 107 50 C3H/HeN, C3H/HeJ (Kline et al., 2014)

UPEC HT7 108 50 C3H/HeN, C3H/HeOuJ (Chassin et al., 2007)

UPEC CP9 6.25 × 105 27 C3H/HeOuJ (Garcia et al., 2013)†

UPEC AL511, AL10 108 50 C3H/HeOuJ,C3H/HeJ,C57BL/6J (Chassin et al., 2006)

UPEC Clinical 108 100 C3H/HeN (Connell et al., 1996)

UPEC UTI89 107 50 C3H/HeN (Kostakioti et al., 2012)

ABU E. coli 83972 2.5 x 108 20 C3H/HeJ (Watts et al., 2012b)

UPEC UTI89, J96, DS17, AAEC185/pSH2

1 × 106 or 1010 10,25 or 100 129/sv (Bates et al., 2004)

UPEC, E. coli Various 109 Albino OF1 (Nielsen et al., 2014)

UPEC, K. pneumoniae

UIT89, TOP52

2 × 107-8

50

C3H/HeN, DBA/2J, BALB/c, C3HeB/FeJ, 129S1/SvImJ, C3H/HeOuJ, C57BL/6J, C3Smn.CB-Prkdcscid/J, CBA/J,

(Hannan et al., 2010)

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C3H/HeJ,

K. pneumoniae, S. saprophyticus

ATCC33459, ATCC15305

105 25 THP gene KO (Raffi et al., 2005)

P. stuartii BE2467 107 50 CBA/J (Armbruster et al., 2014)*

P. stuartii NS 109 NS CD-1, CBA (Johnson et al., 1987)

P. mirabilis 2921 108 50 CD-1 (Umpierrez et al., 2013)

P. mirabilis HI4320 107 50 CBA/J (Armbruster et al., 2014)*

P. mirabilis HI4320 107 50 CBA/J (Pearson et al., 2011)†

P. mirabilis Clinical 105 50 129/sv (Raffi et al., 2009)

P. aeruginosa MPAO1 5 × 106 50 Swiss-Webster (Bala et al., 2014)

UPEC CP9 6-4 × 107-9 25 or 50 Swiss-Webster (Russo et al., 1996b)

UPEC CP9 3-8 × 107 25 or 50 Swiss-Webster (Russo et al., 1996a)

E. faecalis 12030 3-5 × 108 100 BALB/c (Wobser et al., 2014)

GBS NS 103 NS Cornett (Furtado, 1976)

GBS, UPEC 247, CFT073 3 × 109 40 C57BL/6 (Tan et al., 2012a)†

GBS, UPEC 247, CFT073 109 20 C57BL/6 (Ulett et al., 2010)

- CAUTI & Biofilm Formation Studies -

UPEC UTI89 1-2 × 107 50 C57BL/6Ncr (Guiton et al., 2012)

UPEC UTI80 108 50 C3H/HeN, C3H/HeJ (Anderson et al., 2003)

UPEC UTI89 2 × 107 50 C3H/HeN (Anderson et al., 2010)

E. faecalis OG1RF 105-6 200 ICR (Singh et al., 2007)

E. faecalis OG1RF 104 200 ICR (Kemp et al., 2007)

E. faecalis OG1RF 2-3.5 × 107 50 C57BL/6Ncr (Guiton et al., 2010)

E. faecalis OG1RF 1-2 × 107 50 C57BL/6Ncr (Flores-Mireles et al., 2014)

E. faecalis OG1RF 4 × 107 50 C57BL/6 (Nielsen et al., 2012)

P. aeruginosa PA14 106 35 CF-1 (Cole et al., 2014)

P. mirabilis 296 2.5 × 104 50 C57BL/6 (Janssen et al., 2014)

K. pneumoniae TOP52 2.3-3.3 × 107 50 C57BL/6NCr (Murphy et al., 2013)

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- Treatment and Preventative Studies -

UPEC UTI89 108 50 C57BL/6 (Schilling et al., 2002)

UPEC UTI89 108 100 i.p. C57BL/6Ncr (Goller et al., 2014)

UPEC UTI89 1-2 × 107 50 C57BL/6Ncr (Guiton et al., 2012)

UPEC CFT073, 83972 1 × 106 or 108 25 C57BL/6 (Watts et al., 2012a)

UPEC Clinical 8 × 108 80 Ssc-CF1 (Hvidberg et al., 2000)

UPEC Clinical 108 50 BALB/c (Tratselas et al., 2014)

UPEC EC958 2 × 107-8 NS C3H/HeN (Totsika et al., 2013)

UPEC UTI89 1-2 × 107-8 50 C3H/HeN (Hannan et al., 2014)

CT073, HT7 50 C3H/HeN (Vimont et al., 2012)

UPEC UTI89 107 50 CBA/J (Blango and Mulvey, 2010)

UPEC CFT073 108 50 CBA (Lefort et al., 2014)

UPEC 9612 2.2 × 109 50 CD-1 ICR (You et al., 2009)

P. mirabilis Pr2921 2 × 108 50 CD-1 (Scavone et al., 2014)

P. mirabilis Pr2921 2 × 108 50 CD-1 (Pellegrino et al., 2003)

P. aeruginosa SR10396 4.7 × 104 100 SLC/ICR (Tsuji et al., 2003)

*Same study. †Study that reports transcriptomic analysis based on microarrays. NS: Not stated. i.p.: intraperitoneal challenge for

urosepsis model.

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Table 2. Normative standards for murine UTI models and application-based

modifications

Variable Standard Method Alternative Methods

Dose Single dose of 108 cfu 107 cfu in studies using fimbrial-enriched cultures (i.e. serial static subcultures) One repeat (2nd) dose for superinfection

Volume

25-50 µl PBS

Increase volume to 100 µl to model pyelonephritis, induce VUR, or for i.p./i.v. Carrier alternatives: 1% India ink PBS

Inoculum

Shaking cultures

For studies of fimbrial adhesion, serial static subcultures ideal

Injection

Deep transurethral insertion, manual injection

Use of electronic or mechanical injection pumps, mini-surgical via subrapubic delivery or use of indwelling catheter Intra-urethral, or perurethral challenge Use of i.p. delivery 108 cfu for urosepsis Use if i.v. delivery ? cfu for urosepsis Alternative methods as defined by model

Post-challenge

No restrictions on inoculum voiding

Restrictions on inoculum voiding post-challenge, through diet/water restriction, collodion film, or catheter clamping

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Table 3. Future development of murine UTI models for study of human disease

Model What’s needed Possible Outcome/Advancement

Urosepsis Characterization of sepsis/systemic infection models that more closely mimic human route of infection (bladder � kidney � blood)

More physiological studies of human urosepsis Enable novel comparisons of uropathogens

Transurethral infection

Comparison of intra-urethral and/or superficial perurethral challenge approach for injection Standardization of challenge dose and volume Characterization of superinfections models

Identify potential differences between methods Allow a standardized method for more direct comparisons between different studies Model rUTI after frequent sexual intercourse

ABU Study of dynamics of bacterial persistence in urine in the absence of severe inflammation Comparison of urinary constituents between mice and humans utilized by bacteria in ABU

More physiological studies of mechanisms underlying human bacteriuria and ABU Identify bacterial metabolic processes in ABU

CAUTI Development of indwelling catheter models for different uropathogens that cause CAUTI

Study virulence factors for biofilm formation

Pregnancy Characterization of UTI during pregnancy Link consequences of untreated UTI to adverse pregnancy outcomes

SCID-hu UTI Development and characterization of novel humanized murine models of UTI

More physiological studies of human UTI Identify human-specific factors and responses involved in the control of, or susceptibility to, UTI

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Figure 1: Similarities between murine models of UPEC UTI and human infection. 184x117mm (300 x 300 DPI)

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Figure 2: Three different inoculation techniques for murine UTI. 199x138mm (300 x 300 DPI)

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Figure 3: The severity of inflammation and time course of UPEC bladder infections in different mouse strains. 104x183mm (300 x 300 DPI)

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c Consult author(s) regarding copyright matters · 2021. 8. 7. · 53 2009, Hola et al., 2012, Stahlhut et al., 2012), and are often associated with enhanced 54 antibiotic resistance