You might not think much about what's in a baby's diaper, but to scientists, it's a treasure trove of information about human health.
Imagine a bustling city inside a baby's gut—trillions of microscopic inhabitants working together to digest food, train the immune system, and protect against harmful invaders. Among the most important residents are bifidobacteria, particularly Bifidobacterium longum, which often dominates the gut microbiota of infants. What most people don't realize is that this species contains different subspecies that play distinct roles in human health. Until recently, scientists struggled to tell them apart, until a clever genetic detective story unfolded, centered around a surprising gene variation.
The human gut contains approximately 100 trillion microorganisms—more than 10 times the number of human cells in our bodies!
The mystery began when researchers decided to investigate the tuf gene in Bifidobacterium longum subspecies from Chinese infants. What they found challenged assumptions about how we identify these microbes and revealed important insights about the delicate ecosystem that develops in our earliest days of life 1 .
Within the species Bifidobacterium longum, two particular subspecies—B. longum subsp. longum and B. longum subsp. infantis—are known to colonize the infant bowel. Though they sound similar, these subspecies have different capabilities and likely play different roles in human health.
Specialized in utilizing human milk oligosaccharides (HMOs)
More limited HMO utilization capabilities
The most significant difference lies in their ability to process human milk oligosaccharides (HMOs)—complex sugars abundant in breast milk that human infants can't digest on their own. Think of HMOs as specialized fuel that only certain microbes can use. B. infantis is particularly adept at utilizing a wide variety of HMOs, while B. longum has more limited capabilities 1 . This special skill may make B. infantis a keystone species in the infant gut, helping to shape the entire microbial community during a critical window of development.
Key Insight: For years, scientists have faced a challenge: how to quickly and accurately measure the abundance of each subspecies in the complex mix of fecal microbiota. The standard method had been to look at the 16S rRNA gene, but its sequence proved too similar between the subspecies to reliably tell them apart 1 .
Scientists turned their attention to the tuf gene, which codes for a protein called elongation factor Tu. This protein plays a crucial role in protein synthesis—it's essentially a molecular matchmaker that helps assemble the building blocks of proteins in all living cells. Because this gene is essential for survival, it's present in all bacterial cells, but with enough variation between subspecies to potentially serve as a distinguishing marker 1 .
The tuf gene provides instructions for making elongation factor Tu, a protein essential for assembling other proteins in bacterial cells.
The research team developed two specialized TaqMan qPCR assays—highly sensitive tests that could potentially distinguish between the tuf genes of B. longum subsp. infantis and B. longum subsp. longum. When tested on reference strains of known subspecies, the assays worked perfectly, cleanly differentiating between them without cross-reaction with other common bifidobacterial species like B. bifidum and B. breve 1 .
Confident in their new tool, the researchers applied it to the real world: fecal samples from a cohort of Chinese infants. They compared the abundance estimates from their tuf gene method with those from high-throughput sequencing (HTS), expecting the results to align. Instead, they encountered a puzzle—the numbers didn't match 1 .
When the tuf gene qPCR results didn't correlate well with the HTS data (showing a surprisingly low r² value of 0.2127), scientists realized something was wrong. Their carefully designed assays seemed to be missing a substantial portion of B. longum in the fecal samples 1 .
Comparison between expected and actual detection rates of B. longum subspecies using the tuf gene assay.
Researchers found a previously unknown tuf gene variant designated OTU49
In some infants, this variant accounted for 26% of all B. longum sequences
Shared features with both B. infantis and B. longum subsp. longum
The investigation led to the discovery of a previously unknown tuf gene variant, which the researchers designated OTU49 (Operational Taxonomic Unit 49). In some infants, this variant accounted for a remarkable 26% of all B. longum sequences 1 . When they sequenced this mystery variant, they found it shared characteristics with both B. longum subsp. infantis and B. longum subsp. longum—a genetic chimera that didn't fit neatly into either category.
The OTU49 variant matched the B. longum subsp. infantis forward primer, but the B. longum subsp. longum probe and reverse primer, explaining why it had slipped past both of their detection assays 1 . The researchers had found something that challenged their understanding of how to classify these bacteria.
| Method | Target | Advantages | Limitations |
|---|---|---|---|
| 16S rRNA gene sequencing | 16S ribosomal RNA gene | Widely used, standardized | Cannot reliably differentiate between B. longum subspecies |
| tuf gene qPCR (initial assay) | elongation factor Tu gene | Specific discrimination between reference strains | Failed to detect OTU49 variant in real samples |
| High-throughput sequencing | Entire microbial DNA | Comprehensive view of microbiota | Expensive, computationally intensive, not quantitative |
Faced with this puzzling microbe, the research team employed multiple approaches to determine its true identity:
They generated a draft genome sequence for the OTU49 isolate and compared it with available B. longum genome sequences. The analysis revealed high homology and synteny (gene order conservation) with known B. longum subsp. infantis isolates ATCC 15697 and JCM1222. A particularly telling finding was a large inverted genomic region compared to B. longum subsp. longum genomes—a structural signature that aligned OTU49 with the infantis subspecies 1 .
Most importantly, the OTU49 genome contained the HMO utilization region previously identified in B. infantis—the specialized genetic machinery that enables this subspecies to thrive on human milk oligosaccharides 1 .
The researchers then tested how OTU49 grew on different carbohydrate sources compared to reference strains of both subspecies. The results were striking:
| Substrate | B. longum subsp. longum | B. longum subsp. infantis | OTU49 Isolate |
|---|---|---|---|
| Basal medium (no added carbs) | Limited growth | Limited growth | Limited growth |
| Lacto-N-tetraose (LNT) | Strong growth | Strong growth | Strong growth |
| Acidic HMOs | Limited growth | Strong growth | Strong growth |
| Sialic acid | Limited growth | Strong growth | Strong growth |
| Lacto-N-neotetraose (LNnT) | Limited growth | Strong growth | Strong growth |
OTU49 and B. longum subsp. infantis strains grew well on all HMO substrates, while B. longum subsp. longum strains showed robust growth only on LNT 1 . The growth profile clearly grouped OTU49 with the infantis subspecies.
Based on both genomic evidence and functional growth characteristics, the researchers concluded that OTU49 belongs to B. longum subsp. infantis, despite its unusual tuf gene sequence 1 . This discovery explained why the original tuf gene assays had failed—natural genetic variation within the subspecies was greater than previously recognized.
| Tool/Reagent | Function in Research | Application in This Study |
|---|---|---|
| TaqMan qPCR assays | Quantitative measurement of specific DNA sequences | Developed to target tuf genes of B. longum subspecies |
| High-throughput sequencing | Comprehensive analysis of all genetic material in a sample | Revealed total B. longum content, including unexpected variants |
| Simulated digestion models | Replicates human gastrointestinal conditions | Used in related studies on HMO utilization 7 |
| API 50 CH strips | Standardized testing of carbohydrate fermentation | Employed in similar research to profile metabolic capabilities 9 |
| Anaerobic chamber | Creates oxygen-free environment for growing sensitive bacteria | Essential for cultivating bifidobacteria, which are obligate anaerobes |
| Genome assembly software | Pieces together sequenced DNA fragments into complete genomes | Used to reconstruct the OTU49 genome for comparison |
This detective story extends far beyond academic curiosity. The researchers concluded that targeting tuf gene sequences cannot reliably differentiate between B. longum subspecies due to natural sequence variation 1 . Instead, they suggested that functional genes involved in carbohydrate metabolism might be better targets because they directly relate to ecological function 1 .
For scientists studying gut microbiota, this research highlights the importance of choosing genetic targets that reflect functional capabilities rather than relying solely on standard marker genes.
The discovery underscores that genetic diversity exists even within subspecies, and this diversity may represent adaptations to different environments or dietary patterns.
Since B. infantis is uniquely equipped to utilize HMOs, accurately measuring its abundance could help us understand how breast milk shapes the infant gut ecosystem.
Recent studies continue to highlight the importance of B. infantis in gut health. For instance, 2025 research investigated the synbiotic combination of 2'-fucosyllactose (2'-FL, a prominent HMO) with B. infantis EFEL8008, demonstrating how this pair selectively promotes beneficial microbial shifts in adult gut models 7 . This illustrates the ongoing relevance of understanding the specific capabilities of B. infantis strains.
The tale of the tuf gene variation in B. longum subsp. infantis reminds us that nature often defies our neat classification systems. What began as a straightforward effort to improve detection methods turned into a discovery of hidden diversity within a microbe that co-evolved with humans.
As one research team noted, knowledge of the occurrence and abundances of different bifidobacterial subspecies is essential for developing "a concept of the multispecies ecology, especially trophic interactions, of the bifidobacterial population of the infant bowel" 1 . This understanding may ultimately help us manipulate the microbiota to better support infant health—all thanks to careful scientific detective work that began with a puzzling genetic variation.
As we continue to explore the microscopic world within us, each answered question reveals new mysteries waiting to be solved. The humble tuf gene variation detected in Chinese infants represents not just a scientific footnote, but a meaningful step toward understanding the complex partnership between humans and their microbial inhabitants—a relationship that begins in infancy and influences our health for a lifetime.