Most Kidney Stones May Actually Be Bacterial Colonies

THE surgeon’s laser had just finished shattering the kidney stone when something caught Kymora Scotland’s attention. She was examining the fragments under the microscope at UCLA. Fragments that, according to conventional wisdom, should contain nothing but crystals. Instead, she saw something alive.

Layered between the calcium oxalate crystals were bacterial cells, wrapped in the fibrous matrix characteristic of biofilms. Not just scattered bacteria that might have contaminated the sample, but organised communities, embedded deep within the stone’s internal structure. Scotland, a urologist, realised she was looking at something that wasn’t supposed to exist. For decades, calcium-based kidney stones—the kind that afflict roughly 1 in 11 people: have been considered purely chemical formations, sterile aggregates of minerals crystallising from supersaturated urine. The only stones traditionally linked to bacteria were rare struvite stones, comprising just 2 to 6 per cent of cases.

Scotland’s discovery suggests we’ve been fundamentally wrong about how the most common type of kidney stone forms. Calcium oxalate stones make up nearly 80 per cent of all kidney stones. If bacteria are integral to their formation rather than incidental passengers, it changes everything we thought we knew about nephrolithiasis.

The UCLA-led team used an arsenal of advanced imaging techniques (scanning electron microscopy, fluorescent microscopy, focused ion beam sectioning) to peer inside human kidney stones collected during surgery. What they found challenged assumptions held since the field’s inception. In stone after stone, including many from patients without urinary tract infections, bacterial biofilms appeared as intrinsic structural components. The stones weren’t merely colonised on their surfaces; bacteria were intercalated throughout, forming distinct layers between crystalline mineral deposits.

When Scotland and her colleagues polished the stones flat and stained them for DNA, the bacterial organisation became even more striking. Fluorescent bands of DNA lit up in regular stripes, marking bacterial layers with “high regularity in thickness and periodicity,” as the team reports in the Proceedings of the National Academy of Sciences. The bacteria weren’t random interlopers. They were part of the architecture.

The evidence went beyond visual patterns. When the researchers measured crystal sizes in different regions of the stones, they found something revealing about the bacteria’s role. In areas where bacterial biofilms were present, the calcium oxalate crystals averaged just 7 micrometres in diameter. In purely crystalline layers without bacteria, the crystals ballooned to an average of 236 micrometres: more than 30 times larger. The dramatic size difference points to a higher concentration of nucleation sites in bacterial areas, suggesting the microbes actively seed crystal formation rather than simply living amongst already-formed stones.

To confirm the bacteria were real and not just bacterial-shaped artefacts, the team applied chemical stains that selectively bind to components unique to microbial biofilms: lipids from cell membranes, polysaccharides from the protective matrix, and DNA from both cells and the surrounding biofilm structure. Control samples of pure calcium oxalate minerals showed no preferential staining. The human kidney stones lit up.

Perhaps most surprisingly, viable bacteria emerged when stones were cultured. Of 22 stones analysed, 17 harboured living microbes despite many testing culture-negative in standard clinical tests. The bacterial concentrations ranged over six orders of magnitude, and the species roster read like a who’s-who of urinary tract inhabitants: Enterococcus faecalis, Proteus mirabilis, Escherichia coli, Staphylococcus epidermidis. More than 30 per cent of the stones harboured multiple species, suggesting complex microbial ecosystems can establish themselves within the stone environment.

The team, which included researchers from Washington University and the University of Illinois, went further. Using focused ion beam milling (essentially a precision surgical tool that works at the nanoscale) they dissected stones layer by layer under ultrahigh vacuum. Large voids appeared within the crystals, and filling those voids were bacteria and biofilm. The microbes weren’t just on the surface. They were entombed deep inside, embedded during the stone’s growth.

“This breakthrough challenges the long-held assumption that these stones develop solely through chemical and physical processes,” Scotland says. The discovery opens questions about why bacteria would be there at all. Biofilms typically form where bacteria have access to nutrients and can anchor themselves, not inside mineral deposits. Co-author Gerard Wong, who studies the physical chemistry of biological systems at UCLA, suspected the answer might lie in the bacteria’s struggle to survive in urine.

Urine presents a particular challenge for bacteria: it’s loaded with calcium, up to 7 millimolar compared with about 1 millimolar in normal biological fluids. Bacteria must maintain a steep calcium gradient across their membranes (roughly 100 nanomolar inside, 1 millimolar outside) or they die. Pumping out all that extra calcium costs energy. But there’s another strategy: produce extracellular DNA.

When bacteria release DNA into their surrounding biofilm matrix, something interesting happens from a physics perspective. DNA is a highly charged molecule, carrying roughly 5.8 negative charges per nanometre along its sugar-phosphate backbone. That charge density is more than four times greater than synthetic polymers previously studied as potential crystal growth inhibitors. It’s also high enough to trigger what’s called Manning condensation, where positively charged calcium ions are drawn to and “condense” around the negatively charged DNA in a concentrated sheath.

This condensation effectively sequesters calcium ions from the environment, relieving the bacteria of the metabolic burden of constantly pumping them out. But those calcium-dressed DNA molecules then become templates: nucleation sites where calcium oxalate crystals can begin forming. A single bacterial cell, if lysed, releases about 4 megabases of DNA, enough to sequester some 3.6 million divalent calcium ions.

“We found a new mechanism of stone formation that may help to explain why these stones are so common,” Scotland says. Recent studies show that Pseudomonas aeruginosa, a bacterium sometimes found in urinary tract infections, secretes anomalously large amounts of extracellular DNA when cultured in urine. The urine environment itself may be triggering the very behaviour that promotes stone formation.

The findings could explain long-standing clinical puzzles. Why do kidney stones so often recur (up to 80 per cent recurrence in some patient populations)? If bacteria remain viable inside stones, even small fragments left behind after surgical removal could seed new stone growth. Why is there a connection between recurrent urinary tract infections and recurrent kidney stones? The link may be more direct than circumstantial if bacteria are causative agents rather than opportunistic colonists.

Scotland emphasises that bacterial involvement doesn’t mean all kidney stones are “infectious” in the traditional sense. Many patients had no underlying urinary tract infections. The bacteria may be normal inhabitants of the urinary tract that, under the right conditions—high calcium concentration, certain flow dynamics, particular urine chemistry—shift from harmless residents to stone-formation catalysts.

The implications ripple outward. Current treatments for calcium oxalate stones are limited: hydration, dietary changes, surgical removal. Antibiotics are used for struvite stones but not calcium stones, partly because calcium stones have been considered abiotic and partly because antibiotic resistance is a legitimate concern. But if bacteria play a causative role, targeting them (or the biofilms they form) might offer new prevention strategies.

“Our multi-institutional team is currently performing studies to determine how bacteria and calcium-based kidney stones interact,” Scotland says. “We want to understand exactly what makes some patients particularly susceptible to recurrent stone formation, and what it is about these particular species of bacteria that allows them to nucleate these stones.”

One open question is whether the bacteria are the primary cause or secondary contributors. Do they initiate stone formation, or do small crystals form first and then provide surfaces for bacterial attachment, which then accelerates further growth? The intercalated layering suggests cycles of mineral deposition and bacterial colonisation, but the exact sequence remains to be established.

Another frontier is determining whether bacterial biofilms play similar roles in other types of kidney stones. The current study focused on calcium-based stones, but calcium phosphate stones, uric acid stones, and cystine stones also plague patients. If bacteria are endemic to nephrolithiasis rather than specific to calcium oxalate, we may need to reconceptualise kidney stone disease as fundamentally biological rather than purely chemical.

The discovery also resonates with findings in other fields. Neutrophil extracellular traps—networks of DNA that immune cells release to snare pathogens—have recently been implicated in both kidney stone and gallstone formation. Bacterial extracellular DNA, neutrophil extracellular traps: DNA seems to be a recurring theme in pathological mineralisation. The physics is the same whether the DNA comes from bacteria or human cells—highly charged polymers create local environments favourable for crystal nucleation.

For patients, the findings are both sobering and potentially hopeful. Sobering because it suggests kidney stones are more complex than a simple chemical imbalance to be corrected with lemon juice and reduced salt. Hopeful because biological processes are often more amenable to intervention than purely physical ones. Bacteria can be targeted, biofilms can be disrupted, and bacterial behaviours can be influenced.

The research also highlights how much life persists in places we thought were lifeless. An estimated 99 per cent of bacteria can’t be cultured using standard laboratory techniques. They require specific nutrients, complex community interactions, or exist in stressed metabolic states that make them viable but nonculturable. The bacteria inside kidney stones, entombed in crystal, protected by biofilm, surviving on minimal nutrients, represent an extreme of microbial persistence. They were there all along. We just weren’t looking for them.

Scotland’s team examined stones that patients had carried for weeks, months, sometimes years. The biofilms they found were mature, organised, integrated into the stone’s fabric. In some samples, bacteria clustered along crystal cleavage planes, the natural weak points in the mineral structure. In others, they filled voids left by the stone’s polycrystalline growth. Each stone told a story of mineral and microbe co-evolving, layer by layer.

The conventional model of kidney stone formation posits that stones begin when crystals nucleate in supersaturated urine and grow until they become too large to be washed away in the urinary stream. This still happens, likely. But Scotland’s findings suggest there’s often another player in the process: communities of bacteria managing their calcium load by releasing DNA that inadvertently creates the perfect template for the very minerals that will eventually encase them.

It’s a familiar pattern in biology: organisms solving one problem create another. Bacteria produce extracellular DNA to cope with high calcium. The DNA attracts calcium. The calcium precipitates as crystals. The crystals trap the bacteria. The trapped bacteria continue producing DNA. More crystals form. Layer by layer, the stone grows, a monument to unintended consequences.

Study link: https://www.pnas.org/doi/10.1073/pnas.2517066123