Bacteria have their own characteristic genetic profiles. Culture-independent sequencing of bacterial DNA allows characterization of the many bacteria residing in humans. The technology identifies individual bacteria by either sequencing entire bacterial genomes using whole-genome shotgun sequencing (shotgun metagenomics) or by targeting a specific genetic region for sequencing.¹³ The latter typically uses the 16S ribosomal RNA bacterial gene to identify bacteria. The 16S gene has highly conserved regions that are identical in all bacteria as well as variable regions that serve as genetic signatures identifying bacteria to the genus or, in some cases, to the species level.¹³ Both methods have advantages and disadvantages. Shotgun metagenomics may be more costly but may offer greater taxonomic and functional resolution, whereas 16S is less costly and may be used to analyze large numbers of samples.¹⁴
Notably, both methods involve lysis of human cells and neither can distinguish between live and dead bacteria in samples.¹³

The HMP found that the humans harbor bacteria that are characteristic for specific niches.¹⁵,¹⁶ The original HMP did not evaluate the lower urinary tract because urine was believed to be sterile. Sequencing technology has since discovered that although urinary bacterial biomass is relatively sparse, urinary bacterial communities do exist.¹³,¹⁶,¹⁷,¹⁸ Traditionally, cultures were developed to identify Escherichia coli, the most common uropathogen. Recognition of the existence of the urinary microbiome led to development of nontraditional culture techniques identifying a wider range of bacteria, complementing sequence findings. The expanded quantitative urine culture (EQUC) technique cultivates bacteria under aerobic and/or anaerobic conditions with longer incubation, better identifying fastidious organisms.¹⁷,¹⁸ In contrast to gene sequencing, EQUC identifies live bacteria verifying that bacteria found with 16S methods can represent live bacteria in urine, an environment previously considered sterile.¹⁷,¹⁸ EQUC, following bacterial identification with genetic sequencing, better quantifies bacteria in samples and may better identify them to the species level.¹⁷ EQUC disadvantages include an inability to identify all bacteria in samples¹³ and because eukaryotic cells are not lysed during EQUC, intracellular bacterial communities (see following discussion) may remain unrecognized. Nonetheless, nontraditional culturing, genomics, and metabolomics (study of bacterial metabolic products) work in tandem to increase understanding of UTI.

Vaginal microbiome studies illustrate how genomics and metabolomics have altered interpretation of bacterial associations with health and disease. Vaginal Lactobacillus has historically been equated with health, dating back to Döderlein’s 1892 report of a lactic acid producing bacillus (later identified as Lactobacillus) inhibiting pathogen growth.¹⁹ This resulted in the prevalent, although perhaps simplistic view, that Lactobacillus ensures health.¹⁹ Microbiome studies have since found that Lactobacillus dominates vaginal communities in many but not all women and that Lactobacillus nondominant communities can also provide a healthy, acidic environment.²⁰ Understanding differences in the relative abundance and characteristics of Lactobacilli to the Lactobacillus species level provides further insight into their interactions with pathogens. Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii are the most common vaginal Lactobacillus species.¹⁶,²⁰,²¹ Their relative abundance in women differs with age, ethnicity/race, and behaviors (e.g., contraceptive, antibiotic, and hormonal use and sexual activity).¹⁶,²² Lactobacillus species metabolomes also vary. L. iners cannot synthesize D-lactic acid, which is more protective against dysbiosis than the L-lactic acid produced by L. iners.²² L. iners has been associated with vaginal dysbiosis and greater susceptibility to HIV and Chlamydia infections.²² This contrasts with L. crispatus, which synthesizes both D- and L-lactic acid and has been reported to be associated with vaginal stability and health.²² Lactobacilli are also relevant to the urinary microbiome. Presence of Lactobacillus species in the vagina correlates highly with their presence in urine, as demonstrated in a comparative study of 197 paired vaginal and urine samples.²³ Furthermore, in a phase 2 randomized controlled trial of reproductive-aged women with rUTI, L. crispatus provided as a vaginal supplement resulted in decreased rUTI in those who achieved high-level colonization of L. crispatus.²⁴

Lactobacilli developed survival mechanisms beneficial to both host and bacteria, or homeostatic mutualism. In contrast, pathogenic bacteria developed mechanisms detrimental to the host, or bacterial virulence. The most common bacteria responsible for UTI are uropathogenic E. coli (UPEC) followed by Klebsiella pneumoniae, Enterococcus faecalis, Proteus mirabilis, group B Streptococcus, Pseudomonas aeruginosa, Staphylococcus species, and Candida species.¹⁰,²⁵

UTI often occurs when bacteria from nearby niches migrate to the urinary tract, adhere to, and then colonize or destroy host cells.¹⁰,¹⁶,²⁵ Murine and UPEC models have enhanced understanding of bacterial factors associated with human UTI (Fig. 37.1).¹⁰,¹⁶,²⁵ Bacterial pili, commonly found on UPEC, Klebsiella, Proteus, and Enterococcus species, facilitate bacterial migration and mediate adherence to host cells.¹⁰,²⁵ UPEC also invade superficial umbrella cells of mice, forming intracellular bacterial communities.¹⁰,²⁵ Although intracellular communities (in humans and mice) develop acutely, some persist chronically, allowing bacteria to multiply, avoid host surveillance, and serve as bacterial reservoirs in rUTI or asymptomatic bacteriuria.¹⁰,²⁶,²⁷ Bacteria also form biofilms; the adhesive proteins (adhesins) produced by pili form a scaffold for bacterial adherence, acting as an extracellular matrix barrier to the host and antibiotics. UPEC, Proteus, Klebsiella, Enterococcus, and Staphylococcus are all capable of biofilm production enhanced in the presence of foreign bodies, such as urinary catheters.¹⁰,²⁵ A final example of bacterial virulence is production of enzymes (e.g., proteases, urease) and toxins (e.g., cytotoxic necrotizing factor in UPEC, hemolysin in UPEC, Proteus species) that lyse host cells, releasing nutrients in the nutrient-poor bladder, enhancing bacterial survival.¹⁰ These capabilities are not limited to pathogens. Lactobacilli are also capable of cell adherence; biofilm formation; and hydrogen peroxide, lactic acid, and bacteriocin production, capabilities that are antagonistic to other bacteria and beneficial to the host.²⁸,²⁹,³⁰,³¹

Host mechanisms preventing UTI include urinary turbulence accompanying voiding (impeding bacterial passage to the bladder), presence of bladder umbrella cells (mucosal barriers to bacterial invasion), and host immune responses aiding bacterial destruction.²⁶ UTI occurs when host defenses are breached. Epidemiologic studies indicate that host susceptibility to rUTI increases with a family history of UTI, age, menopause, spermicide use, and sexual activity.³,²⁵ Urinary microbiome studies in older women have increased understanding of genetic influences in rUTI. A microbiome study in twins found that almost onethird of the bacteria present in twin urinary microbiomes were attributable to genetic predisposition.³² Escherichia and certain Lactobacillus species exhibited particularly high heritability in these subjects. Increased heritability of specific bacteria suggests human genetics influence the urinary microbiome, predisposing women to rUTI.³²

Last, host immunity affects rUTI. Murine models offer insight into the molecular basis of this susceptibility. Transurethral inoculation of UPEC in susceptible mice results either in spontaneous resolution within 2 weeks or development of chronic cystitis. Mice that more commonly develop rUTI more commonly express cyclooxygenase 2, suggesting that specific immunologic reactions affect UTI recurrence.²⁵ Chronic cystitis is also associated with prolonged tumor necrosis factor signaling, suggesting that prior UTI may increase host susceptibility to future infection.²⁵ On a cellular level, chronic cystitis likely occurs by infection-induced monocyte recruitment, followed by exfoliation of superficial bladder umbrella cells (Fig. 37.2). This initially decreases bacterial load but exposes underlying transitional epithelium
to bacterial invasion and development of intracellular bacterial communities.²⁵ Although these mechanisms have yet to be fully validated in humans, women with UPEC rUTI exhibit increased inflammatory biomarkers and bladder histology similar to that of mice with chronic cystitis, suggesting that maladaptive inflammatory reactions contribute to rUTI in humans.²⁵ Interplay between bacteria, their communities, surrounding niches, and the host result in UTI resistance or susceptibility.¹⁶,²⁵ Future studies will likely continue to explore the yet-to-be-identified “core” urinary, vaginal, and gut microbiomes; the interactions between these neighboring microbiomes; and their metabolic profiles.