The Basics of Bacteriuria: Strategies of Microbes for Persistence in Urine

Bacteriuria, the presence of bacteria in urine, is associated with asymptomatic, as well as symptomatic, urinary tract infection (UTI). Bacteriuria underpins some of the dynamics of microbial colonization of the urinary tract, and probably impacts the progression and persistence of infection in some individuals. Recent molecular discoveries in vitro have elucidated how some key bacterial traits can enable organisms to survive and grow in human urine as a means of microbial fitness adaptation for UTI. Several microbial characteristics that confer bacteruric potential have been identified including de novo synthesis of guanine, relative resistance to D-serine, and catabolism of malic acid. Microbial characteristics such as these are increasingly being defined through the use of synthetic human urine (SHU) in vitro as a model to mimic the in vivo environment that bacteria encounter in the bladder. There is considerable variation in the SHU model systems that have been used to study bacteriuria to date, and this influences the utility of these models. In this review, we discuss recent advances in our understanding of bacteruric potential with a focus on the specific mechanisms underlying traits that promote the growth of bacteria in urine. We also review the application of SHU in research studies modeling UTI and discuss the chemical makeup, and benefits and limitations that are encountered in utilizing SHU to study bacterial growth in urine in vitro.


INTRODUCTION: BACTERIURIA AND URINARY TRACT INFECTION
"Asymptomatic Bacteriuria" (ABU or ASB) is synonymous with asymptomatic Urinary Tract Infection (UTI) in defining the isolation of a specified semi-quantitative count of bacteria in an appropriately collected urine specimen from a person without signs or symptoms related to UTI (Rubin et al., 1992;Nicolle et al., 2005). Bacteriuria is a marker for symptomatic UTI (sUTI) and assists in grading the severity of infection. Establishment of bacteriuria depends on entry of an organism with bacteruric potential into the urinary tract and can persist for months or years. Bacteruric potential encompasses microbial survival, growth, and re-growth in urine, and endurance of host defense mechanisms including dilution, voiding, frequent flushing (O'grady and Cattell, 1966a,b), and antimicrobial constituents such as Tamm-Horsfall glycoprotein (aka uromodulin), and P blood group antigen (Hand et al., 1971;Lomberg et al., 1983;Bates et al., 2004). Bacteruric potential can thus influence the persistence of ABU and sUTI. Here, we analyze the differences in microbial strategies used for growth in urine and the methods for modeling bacteriuria using synthetic human urine (SHU).

MICROBIAL BACTERURIC POTENTIAL AND HOST DYNAMICS
Recent reviews have focused on the pathogenesis of acute sUTI (Nielubowicz and Mobley, 2010;Hannan et al., 2012;Ulett et al., 2013), and ABU (Ipe et al., 2013;Schneeberger et al., 2014) and will not be revisited here. We will focus on bacteriuria specifically; the progression of which depends on microbe traits as well as host factors. Most individuals who suffer persistent UTI do not harbor the same strain of organism over time (Hooton et al., 2000), implying that turnover of causal organisms is dynamic. Replacement of colonizing strains during bacteriuria has been studied for Escherichia coli, the most common cause of ABU (Ipe et al., 2013). Long-term bacteriuria appears to select for attenuated virulence phenotypes of colonizing strains (Salvador et al., 2012). While most microbes are killed by urine, different organisms have distinct bacteruric potential. Several traits can affect microbial growth in urine. Antibiotics stop the progression of bacteriuria (Schneeberger et al., 2012) but patients infected with E. coli experience re-colonization with the same or similar organism at high rates (Dalal et al., 2009). This highlights the dynamic nature of bacteriuria and the role of therapeutic intervention [that is not recommended as routine for ABU (Nicolle, 2014)]. Other factors that are associated with the promotion of long-term bacteriuria are defects in immune signaling pathways such as TLRs (Ragnarsdóttir and Svanborg, 2012). Thus, persistence of bacteriuria relates to microbial bacteruric potential and host characteristics/dynamics including genetic immunodeficiency, re-current infection or strain replacement, and antibiotic therapy.

Microbial Metabolism and Growth Fitness in Urine: Knowledge from E. coli
The progression of bacteriuria depends on a microbes' ability to survive the antimicrobial properties of urine. Urine survival and growth maintains a pool of colonizing organisms regardless of adherence to host cells, and urodynamic properties (urine flow rates, voiding) that differ between individuals (Wullt et al., 1998). Non-voided organisms in residual urine can grow and re-grow to maintain infection. Discoveries using ABU microbes have shaped our understanding of how bacteriuria progresses. ABU E. coli strain 83972 displays robust fitness for urine growth (Klemm et al., 2006;Roos et al., 2006b) though this is not a defining feature of all ABU E. coli, and is observed in some uropathogenic E. coli (UPEC) (Stamey and Mihara, 1980;Alteri and Mobley, 2007;Alteri et al., 2009;Aubron et al., 2012). Poor urine growth has been reported for some fecal E. coli isolates (Stamey and Mihara, 1980;Gordon and Riley, 1992). ABU E. coli 83972 has been investigated as a prophylactic means to treat acute sUTI (Hull et al., 2000;Wullt, 2003;Roos et al., 2006a;Sundén et al., 2006;Klemm et al., 2007;Watts et al., 2012a). The metabolic basis for urine growth of ABU E. coli 83972 involves transport and degradation pathways for galacturonate, glucuronide and galactonate (Roos et al., 2006b), and antioxidant defense mechanisms (Aubron et al., 2012); the details are described elsewhere (Roos et al., 2006a,b). More recently, analysis of ABU E. coli 83972 re-isolates indicated marked versatility of metabolic pathways in urine, including utilization of amino acids, hexuronates or (deoxy-) ribonucleosides as an adaptation to individual hosts (Zdziarski et al., 2010). This underlines the metabolic versatility of E. coli in urine in response to hostspecific metabolic constraints. guaA and argC were shown to be critical for urine growth and a lack of urinary guanine (or derivatives), combined with an inability of E. coli to synthesize these compounds de novo, prevents the synthesis of other guanine (or derivative)-dependent products that are required for growth (Russo et al., 1996). Separate from guanine, argC and carAB mutants had reduced growth in urine in a E. coli transposon mutagenesis study, illustrating a role for arginine metabolism (Vejborg et al., 2012).

Knowledge from Bacteria other than E. coli
Clinical isolates of Enterococcus faecalis, Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus saprophyticus, and Streptococcus agalactiae have been shown to grow in human urine (Table 1). However, little is known about the mechanisms used by these organisms for urine growth. For E. faecalis there are some similarities to E. coli; E. faecalis expresses multiple virulence genes in urine (Shepard and Gilmore, 2002) including genes for iron transport (Vebø et al., 2010). Iron utilization mechanisms have been reported in urine growth assays with E. coli (Watts et al., 2012b). Limiting manganese may be important in restricting E. faecalis urine growth (Järvisalo et al., 1992;Low et al., 2003;Vebø et al., 2010). In contrast to activation of pathways described for E. coli, E. faecalis activates citrate and aspartate metabolic pathways, and represses glucose uptake (Vebø et al., 2010). Human urine contains more citrate than glucose (Shaykhutdinov et al., 2009;Wishart et al., 2009;Bouatra et al., 2013), which could promote growth of E. faecalis. However, human urine is highly variable in chemical constituency and different levels of components such as glucose may influence microbial growth; for example, glucosuria enhances growth of E. coli. E. faecalis also upregulates genes for utilization of sucrose (and perhaps fructose), another constituent of urine (Tasevska et al., 2005;Bouatra et al., 2013). Other E. faecalis genes thought to function in urine growth include those related to import of phosphorylated sugars and glycerol, N-acetyl glucosamine metabolism (Vebø et al., 2010), cysteine synthase, and pathways for conversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate. Urinary aspartate may be used for nitrogen metabolism (Guo and Li, 2009). E. faecalis is auxotrophic for multiple amino acids but human urine contains several amino acids including arginine, glutamate, glycine, and leucine (Guo and Li, 2009;Bouatra et al., 2013).
An analysis of metabolic traits of P. aeruginosa using synthetic urine revealed adaptation in central metabolism to lactate, citrate, and amino acids as carbon sources, and the induction of amino acid utilization pathways (Tielen et al., 2013). Metabolic flux analysis showed the use of the Entner-Doudoroff pathway with respiratory metabolism, with the pentose phosphate pathway being used exclusively for biosynthesis. Flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, and likely caused by adaptive processes in individual strains during infection (Berger et al., 2014).

Trait that confers bacteruric potential Bacterial species References
Ability to utilize human urine as a substrate for growth E. coli Gordon and Riley, 1992;Russo et al., 1996;Roesch et al., 2003;Snyder et al., 2004;Johnson et al., 2006;Roos et al., 2006a;Alteri and Mobley, 2007;Klemm et al., 2007;Aubron et al., 2012;Vejborg et al., 2012;Watts et al., 2012b;Hryckowian et al., 2015;King et al., 2015 Phenotype metabolic arrays were recently reported for ABU S. agalactiae (Ipe et al., 2016). Comparison with S. agalactiae strains that were unable to grow in urine showed that malic acid catabolism was important for growth in urine. Malic acid utilization is related to the malic enzyme metabolic pathway that catalyzes the oxidative decarboxylation of malate (a component of human urine depending on diet) to pyruvate and CO 2 . This is related to malolactic fermentation, a bacterial metabolic process typically associated with wine deacidification (Landete et al., 2010).
Three Ways to Survive in Urine: Resistance, Acquisition, and Osmoadaption Urine is naturally antimicrobial; hypertonicity with low pH (averaging ∼6.0) and high concentrations of urea inhibit most bacteria (Chambers and Lever, 1996;Kucheria et al., 2005). Nitrite in mildly acidified urine inhibits the growth of some uropathogens (Carlsson et al., 2001), and other abundant proteins such as Tamm-Horsfall glycoprotein are antimicrobial (Raffi et al., 2005;Säemann et al., 2005). Recently, the antimicrobial properties of urine were shown to include specific inhibition of both expression and function of UPEC type 1 pili (Greene et al., 2015), and downregulation of capsule genes (King et al., 2015). Individualistic urinary chemical features can also affect the antimicrobial properties of some antimicrobial urinary proteins such as siderocalin (Shields-Cutler et al., 2015). Bacteria that can endure urine antimicrobial properties do so through various means; for example, S. saprophyticus tolerates high concentrations of D-serine, which is abundant in urine and bacteriostatic toward organisms that lack a D-serine deaminase (Cosloy and McFall, 1973;Hryckowian et al., 2015). Iron limitation and the presence iron chelators such as lactoferrin (Weinberg, 1978) compounds the problem of nutritional immunity for microbes (Hood and Skaar, 2012) especially during states of increased iron chelator production (Gonzalez-Chavez et al., 2009;Soler-García et al., 2009). Siderophores confer bacteruric potential to E. coli (Håversen et al., 2000;Snyder et al., 2004;Alteri and Mobley, 2007;Hagan and Mobley, 2009;Garcia et al., 2011;Watts et al., 2012b) and several genes encoding factors involved in iron transport are upregulated in E. faecalis during urine growth (Vebø et al., 2010). Siderophores may also act as ligands for cations other than iron, such as copper (Chaturvedi et al., 2012). Variable resistance to urinary defense molecules (e.g., nitrite, ascorbic acid) could influence bacteruric potential but little is known beyond the effects of these toward the chemical properties of urine (Carlsson et al., 2001). Finally, many organisms are probably unable to survive the oxygen concentrations encountered in the bladder (Leonhardt and Landes, 1963;Clarke et al., 1985).
Urea is abundant in urine and is antibacterial (Sobel, 1985). Osmoadaptive systems enable some bacteria to survive the stressful hypertonic conditions of urine. Bacterial accumulation of osmotically compatible solutes that are present in urine (e.g., betaines) confers bacteruric potential by effectively enabling microbes to resist dehydration (Chambers and Lever, 1996;Deutch et al., 2006). Osmoadaptive systems respond to changes in tonicity in urine, and support survival by counteracting low pH, high urea concentrations and hypertonicity (Chambers and Lever, 1996). For E. coli, glycine betaine is a central osmoprotectant to resist urea toxicity, and its accumulation is essential to adaptive responses to osmotic stress (Kunin et al., 1992;Chambers and Lever, 1996). E. coli increases the activity of potassium transport systems that encompass TrkG, TrkH, and Kup (Meury et al., 1985), or Kdp (Laimins et al., 1978) to counteract osmotic stress. Other systems involve trehalose as an organic osmolyte; its induction is triggered in conditions of high potassium (and glutamate) (Strøm and Kaasen, 1993), and its accumulation elicits the release of potassium from the cell (Dinnbier et al., 1988). This might aid growth since trehalose can be used as a carbon source (Gutierrez et al., 1989;Styrvold and Strøm, 1991). OmpR regulates osmoadaptive genes in E. coli (Barron et al., 1986). Osmotic stress suppresses the expression of fimbriae and flagellin (Kunin et al., 1994(Kunin et al., , 1995. Future studies of bacteria other than E. coli that can grow in urine will elucidate additional mechanisms of resistance to urinary antimicrobial properties, nutrient acquisition and metabolism, and osmoadaption.

Between a Rock and a Hard-Place: The Bladder Mucosa-Lumen Interface of Immune Surveillance and Bacteriuria
Inflammation is a critical part of sUTI pathogenesis (Hannan et al., 2012;Ulett et al., 2013) and involves thousands of genes that drive antibacterial responses within hours of infection (Duell et al., 2012;Tan et al., 2012;Carey et al., 2016). For example, antimicrobial peptides produced by the bladder are important for protection against infection (Chromek et al., 2006). Microbes must survive these inflammatory events. Urine is a "Hard-place" for microbes to survive, as discussed above. Tissue inflammation in the bladder represents a "rock" of antimicrobial responses for defense against UTI and, for ABU, can encompass pyuria, cytokine release (IL-1α, -6, and -8), and antibody production, which has been documented in elderly adults, as reviewed elsewhere (Nicolle, 1997). Excessive inflammation may contribute to chronic sUTI (Hannan et al., 2010) and some acute sUTI symptoms have been linked to specific inflammatory events (Rudick et al., 2010). However, the benign, minimally inflammatory nature of ABU is reflected in the lack of morbidity in individuals who do not receive therapy (Nicolle, 1997(Nicolle, , 1999Ariathianto, 2011). Details of how ABU bacteria induce and minimize inflammation offer insight into how microbial modulation of host defense may promote bacteriuria. ABU E. coli minimizes inflammation by averting adherence due to a lack of fimbriae expression; this limits immune activation (Roos et al., 2006b) and results in long-term ABU (Arthur et al., 1989;Andersson et al., 1991). Grönberg-Hernandez et al. showed that ABU E. coli activates IRF3 and TLR4-dependent signaling, however, triggering a response that depends on host genetic background (Grönberg-Hernández et al., 2011); the IRF3-dependent signaling pathway is critical for distinguishing pathogens from the normal flora (Fischer et al., 2010). TLR4 senses P-fimbriated E. coli (Frendéus et al., 2001), and TLR4 mutations may favor ABU by impeding innate responses . This raises the question of whether ABU may influence subsequent encounter(s) with other uropathogens. One study on streptococcal UTI showed an influence on the severity of subsequent E. coli UTI in mice (Kline et al., 2012). Thus, immune activation triggered by ABU might affect subsequent sUTI caused by diverse pathogens. These data offer some parallel to clinical observations that patients with E. coli ABU suffer re-colonization at high rates following therapy (Dalal et al., 2009).

MODELING BACTERIURIA IN VITRO: SYNTHETIC HUMAN URINE (SHU)
Urine is unique from a microbial perspective and its chemical makeup, distinct from all other bodily fluids, has been modeled for studying the growth of microbes for 50 years (O'grady and Pennington, 1966). Urine has a low pH and a high osmolality due to the presence of salts and urea (Kucheria et al., 2005;Sheewin, 2011). The peptides, proteins, and organic acids present in urine may be metabolized by microbes (Decramer et al., 2008). Urine is dynamic in flow rate and composition, which changes subject to diet, age, gender and health status, and disease. Decreased levels of THP, for example, are associated with diabetes (Torffvit and Agardh, 1993) and infection (Ronald and Ludwig, 2001). Data on microbial traits that afford bacteruric potential have, in many cases, been derived from studies using SHU. Eight original SHU media recipes were described between 1971 and 2010: as summarized according to research application and composition in Table 2.
SHU offers several advantages compared to normal human urine collected from healthy adults for research assays ex vivo; it avoids the issue of variable chemical composition encountered with fresh human urine; variation in urinary constituents between individuals (Bouatra et al., 2013) is a challenge for standardizing research studies. Methods for "normalizing" fresh human urine include pooling samples and adjusting dilution/concentration according to creatinine concentration, specific gravity, and osmolality. The most widely used method is creatinine adjustment (Barr et al., 2005), however no bacteriuria research studies to date have applied these methods for normalizing, and the effects on data interpretation are unknown. Volume limits have been difficult for some studies (Davis et al., 1982). As a surrogate model, the benefits of SHU are defined by how closely it can reflect the chemical complexity of fresh human urine. Urine from a healthy adult contains glucose (0.2-0.6 mM) (Shaykhutdinov et al., 2009), creatine (0.38-55.6 mM; Barr et al., 2005;Shaykhutdinov et al., 2009), and glycine with low levels of other amino acids such as D-serine (Huang et al., 1998;Pätzold et al., 2005), histidine, glutamine, methionine, proline, glutamate, arginine, cysteine, and branched chain amino acids (Guo and Li, 2009;Vebø et al., 2010). It contains trace fatty acids, citrate (1.0-2.0 mM) (Wishart et al., 2009), sucrose (70-200 µM) (Tasevska et al., 2005), and manganese (nM range) (Järvisalo et al., 1992).

CONCLUSIONS AND FUTURE DIRECTIONS
A capacity of microorganisms for urine growth may aid in establishing long-term bacteriuria and is relevant to many microbial species. New discoveries on immune activation by ABU show this form of infection does not exist entirely under the radar of immune surveillance. Continued use of SHU for in vitro studies will drive new discoveries on how bacteriuria progresses and how this may influence subsequent infection. Future work needs to address multiple areas; including (1) validation of the proposed composite SHU medium using a range of relevant bacteria; (2) defining differences in "significant" bacteriuria for different organisms and the implications of bacteruric potential toward such definitions; (3) lifestyle adaptations, other than those described for D-serine, guanine, malic acid, and iron acquisition that aid microbial bacteruric potential; (4) the molecular basis of urine growth in non-E. coli organisms; how ABU interfaces with host immune mechanisms for different organisms (and among distinct patient populations); (5) differences in male vs. female urine composition, in particular hormones (and whether this influences bacterial growth); and (6) how ABU impacts on subsequent UTI including comparisons of immune responses to ABU caused by organisms other than E. coli. How effective ABU might be as a prophylactic approach against sUTI continues to be a topic for future investigation. More importantly, studies aimed at defining bacterial mechanisms that are critical for growth in urine are essential in the context of providing a foundation for novel treatment and preventive strategies.

AUTHOR CONTRIBUTIONS
DI, EH, and GU conceived of the study, analyzed the literature, and wrote the manuscript.