The research team, led by scientists at Shahid Beheshti University of Medical Sciences in collaboration with Hakim Children’s Hospital, collected 79 Shigella isolates from the stool of pediatric patients suffering from diarrhea between September 2023 and April 2025. The children had all been referred to the hospital, one of Tehran’s major pediatric centers, making the sample a representative snapshot of clinically significant infections in the city’s young population. The study was approved by the ethics committee of Shahid Beheshti University of Medical Sciences and funded through a university grant.

Species identification revealed a familiar but shifting landscape. Shigella flexneri remained the predominant species, accounting for 59.5 percent of isolates, while Shigella sonnei made up the remaining 40.5 percent. This distribution matters because the two species differ not only in their epidemiology but also, as the study demonstrated, in their genetic armamentaria and their susceptibility to antibiotics. Globally, S. flexneri has traditionally dominated in low- and middle-income countries while S. sonnei has been more common in industrialized settings, so the near-even split observed in Tehran provides a useful epidemiological marker for a region in epidemiological transition.

The antimicrobial susceptibility testing, performed using the Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standards Institute guidelines, tested isolates against twelve antibiotics. The results were grim. Resistance to ampicillin, a long-standing first-line option, stood at 92.4 percent. Trimethoprim-sulfamethoxazole, another mainstay of treatment in many settings, fared little better, with 91.1 percent of isolates resistant. Tetracycline resistance reached 86.1 percent. These three drugs, which in earlier decades formed the backbone of shigellosis therapy, are now effectively useless against the vast majority of circulating strains.

Not all news was bleak. The majority of isolates remained susceptible to meropenem, a broad-spectrum carbapenem, at 84.8 percent, and to tigecycline, a glycylcycline often held in reserve, at 82.3 percent. These agents thus retain considerable activity against Shigella in this setting, although clinicians must weigh the appropriateness of deploying such broad-spectrum drugs in children, particularly given concerns about preserving these last-line options. More troubling was the species-specific pattern that emerged for other drug classes: S. sonnei isolates demonstrated significantly greater resistance to azithromycin and to the fluoroquinolones than S. flexneri, a difference the authors flagged as statistically significant. Azithromycin and fluoroquinolones such as ciprofloxacin are among the most important oral options for treating shigellosis, so their erosion in S. sonnei narrows the therapeutic window considerably.

Beyond resistance phenotypes, the heart of the study lay in its molecular analysis. The researchers used polymerase chain reaction to screen every isolate for a panel of eleven virulence-related genes, including ipaH, ipaA through ipaD, ipgD, sen, virA, virB, virF, ial, sigA, icsA, and icsB. Many of these genes encode components or effectors of the type III secretion system, a syringe-like molecular apparatus that Shigella uses to inject effector proteins directly into human intestinal epithelial cells. This injection machinery allows the bacterium to invade host cells, escape immune detection, manipulate cell signaling, and spread from cell to cell, producing the colitis and bloody diarrhea characteristic of shigellosis.

The PCR results revealed a strikingly conserved virulence core. Every single one of the 79 isolates carried ipaH, a multicopy gene that is frequently used as a diagnostic target precisely because of its ubiquity and its presence in multiple copies on both the large virulence plasmid and the chromosome. More than 90 percent of isolates also harbored ipaB, ipaC, and ipaD, the genes encoding the invasion plasmid antigens that form the translocation pore and the effector proteins that trigger host cell uptake. The near-universal presence of this core set underscores that the invasive capacity of Shigella remains intact even as the bacterium accumulates resistance determinants; there is no apparent trade-off in which the bacteria sacrifice virulence for resistance.

Not every gene, however, was evenly distributed. The presence of ipgD, which encodes another type III secretion system effector involved in invasion, and sigA, which encodes an enterotoxin contributing to fluid secretion and tissue inflammation, differed significantly between the two species. These species-specific differences in virulence gene carriage suggest that S. flexneri and S. sonnei circulating in Tehran may cause disease through subtly different molecular mechanisms, and they complicate any one-size-fits-all approach to vaccine design or virulence-based diagnostics. A vaccine or therapeutic strategy targeting a single effector might protect against one species but leave the other untouched.

Perhaps the most conceptually significant finding involved the statistical associations between specific virulence genes and resistance phenotypes. The researchers found that resistance to levofloxacin, a fluoroquinolone, was linked to the presence of ipaB, while resistance to ampicillin was associated with ipaD. Even more broadly, sigA was associated with resistance across multiple drug classes. The authors interpret such correlations as evidence that virulence gene profiles could serve as indirect markers of resistance behavior, and they hint at underlying biological or epidemiological mechanisms. Plasmids carrying virulence genes can also carry resistance determinants, and particular genetic lineages that have succeeded in circulating may have acquired both traits together. Co-selection, in which the presence of one trait facilitates the maintenance of another on the same mobile element, is a well-established driver of resistance spread in enteric bacteria, and these associations are consistent with that framework.

The practical implications of the study extend in several directions. For clinicians in Tehran and comparable settings, the near-total loss of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline as treatment options means that empirical therapy must lean increasingly on carbapenems, tigecycline, or whichever oral agents retain activity, decisions that should ideally be informed by species identification and susceptibility testing. For public health authorities, the distinct resistance and virulence profiles of S. flexneri and S. sonnei argue for species-specific surveillance programs that track the two organisms separately rather than lumping them together as generic Shigella. And for researchers, the demonstrated links between virulence genes and resistance phenotypes open a line of inquiry into whether molecular screening for genes such as sigA, ipaB, or ipaD could help predict resistance patterns before culture-based results become available. As the authors conclude, the findings underscore a need for vigilance: in this Tehran pediatric population, shigellosis has become not only a disease of poverty and sanitation but a showcase for how bacterial virulence and antibiotic resistance evolve together in a clinical setting.

Subject of Research: Virulence gene distribution and antimicrobial resistance in pediatric Shigella isolates from Tehran, Iran

Article Title: Distribution of virulence-related genes in Shigella isolates recovered from pediatric patients in Tehran, Iran: association with antimicrobial resistance

Article References: Kazemzadeh Anari, R., Sadredinamin, M., Hashemi, A., Houri, H., Taheri, M., Yousefi Nojookambari, N., Nikmanesh, A., & Ghalavand, Z. (2026). Distribution of virulence-related genes in Shigella isolates recovered from pediatric patients in Tehran, Iran: association with antimicrobial resistance. Gut Pathogens. https://doi.org/10.1186/s13099-026-00875-9

Image Credits: AI Generated

DOI: 10.1186/s13099-026-00875-9

Keywords: Shigella, antimicrobial resistance, multidrug resistance, Shigella flexneri, Shigella sonnei, virulence genes, children, diarrhea, Tehran, type III secretion system, pediatric infectious disease, public health

The research, led by Ethan Gough and Soumya Basu of the Department of International Health at the Johns Hopkins Bloomberg School of Public Health, together with senior author Subhra Chakraborty and colleagues, drew on stool samples and immune measurements from 27 adult volunteers who participated in a controlled clinical trial. Each volunteer received two doses of ACE527, a live attenuated oral ETEC vaccine candidate, and was subsequently challenged with the virulent ETEC strain H10407, a well-characterized isolate that has long served as a benchmark strain in human challenge studies of enteric vaccines. The trial was registered under ClinicalTrials.gov identifier NCT01060748, and the microbiome analysis was approved by the Johns Hopkins University Institutional Review Board under protocol IRB 20100221.

To characterize the bacterial communities living in the participants’ intestines, the team performed 16S ribosomal RNA sequencing on stool samples collected during the trial. This technique amplifies and sequences a molecular marker present in all bacteria, allowing researchers to identify which microbial taxa are present and in what relative abundances, without needing to culture the organisms in the laboratory. From these data the investigators computed two standard measures of community structure: alpha diversity, which captures how many species are present and how evenly distributed they are within a single sample, and beta diversity, which measures how similar or different microbial communities are between individuals or across time points.

The immune outcomes of interest were drawn from enzyme-linked immunosorbent assay measurements of antibody responses directed against two key ETEC antigens: the B subunit of heat-labile toxin, known as LTB, and colonization factor antigen I, or CFA/I, a surface structure the bacteria use to adhere to the intestinal lining. Antibodies were quantified both in serum, reflecting systemic immunity, and in the antibody-in-lymphocyte-supernatant assay, or ALS, which captures antibodies secreted by circulating plasmablasts and serves as a surrogate for mucosal immune activation in the gut. Both IgG and IgA classes were measured, with IgA of particular interest because it is the dominant antibody isotype at mucosal surfaces and is widely regarded as a correlate of protection against intestinal pathogens.

The statistical analysis went well beyond simple comparisons. The team used regression models to link individual bacterial taxa to immune readouts, applied the Microbiome Regression-Based Kernel Association Test, known as MiRKAT, to test whether overall community composition predicted immune responses, and employed a relaxed least absolute shrinkage and selection operator, or LASSO, approach to identify parsimonious sets of taxa associated with protection. Protection itself was assessed clinically, based on whether volunteers developed severe diarrhea and on whether ETEC could be detected as colonizing the gut after the challenge with H10407.

One of the clearest signals emerged from the alpha diversity analysis. Volunteers with higher inverse-Simpson diversity, a metric that increases both with the number of species present and with their evenness, tended to mount weaker ALS IgA responses against both LTB and CFA/I. In other words, a more diverse gut community was associated with a blunted mucosal antibody response to the vaccine. This finding is intriguing because it runs counter to the common assumption that greater microbial diversity is inherently beneficial; in the specific context of oral live attenuated vaccine immunogenicity, a densely populated and diverse intestinal ecosystem may compete with the vaccine strain or modulate innate immune signaling in ways that dampen the antigen-specific response.

Beta diversity told a different story. The overall composition of the gut community, considered as a whole, correlated with increased serum anti-CFA/I IgA, suggesting that the identity of the microbes present, rather than simply their number, shapes the systemic arm of the antibody response. At the level of individual taxa, the vaccination series was associated with a measurable increase, on the order of 25 to 30 percent, in the relative abundance of several groups, including the Eubacterium brachy group, members of Family XIII AD3011, and Actinomyces. Whether these shifts represent a direct effect of vaccine colonization on the ecosystem or an indirect consequence of the immune activation that vaccination triggers remains an open question, but the consistency of the direction of change across participants suggests a reproducible vaccine-microbiome interaction.

Perhaps the most consequential findings concerned specific bacterial families whose abundance tracked with protection. Members of Anaerovoraceae, Peptostreptococcaceae, Oscillospiraceae, and Veillonellaceae were associated with enhanced immune responses and, importantly, with protection against severe diarrhea and against ETEC colonization following the challenge. In contrast, several other groups, including Ruminococcaceae, Sutterellaceae, Coriobacteria, Clostridia, and Actinobacteria, showed antagonistic associations, being linked in the opposite direction with immune outcomes or protection. These antagonistic taxa are not necessarily harmful in their own right; the associations are statistical relationships that require mechanistic follow-up. Nonetheless, the pattern suggests that the interplay between a person’s resident microbiota and an oral vaccine is not uniform across the microbial community but instead involves specific lineages that either support or hinder the generation of protective immunity.

The implications for vaccine development are considerable. Oral live attenuated vaccines must survive passage through the stomach, establish limited replication in the intestine, and present their antigens to the gut-associated lymphoid tissue, and every one of those steps occurs in the context of a complex microbial ecosystem that can facilitate or obstruct them. If the microbiome modulates take of the vaccine strain, the magnitude of mucosal IgA responses, or the likelihood of sterilizing protection after exposure to wild-type bacteria, then microbiome status may help explain a long-standing puzzle in ETEC vaccine research: why candidate vaccines that perform reasonably well in controlled human challenge studies among adults in industrialized settings often show weaker immunogenicity and efficacy when tested in infants and young children in endemic countries, whose gut communities differ markedly from those of the trial volunteers in Baltimore.

The authors emphasize that the study, funded by the National Institute of Allergy and Infectious Diseases through grants R01AI153399 and R56AI168316 and additionally supported by the PATH agreement GAT.1371-05689-CTA, is an early step in a longer research program. With 27 participants, the analysis is necessarily exploratory, and associations identified through sequencing and regression modeling will need to be validated in larger and more diverse cohorts, ideally including populations in ETEC-endemic regions where the public health burden is greatest. Still, the work offers a concrete roadmap: specific taxonomic markers, measurable by inexpensive 16S sequencing, could eventually help researchers stratify volunteers in vaccine trials, stratify risk in endemic populations, or even guide the rational use of microbiome-directed adjuncts such as targeted prebiotics to maximize the protective potential of future ETEC vaccines. As enteric vaccine science matures, it appears increasingly clear that the answer to whether a vaccine works may lie not only in the vaccine itself but in the trillions of microbes with which it shares the intestine.

Subject of Research: How gut microbiota composition influences immune responses and protection following oral ETEC vaccination and challenge

Article Title: Influence of gut microbiota on immune responses and protection in volunteers receiving the live attenuated oral ETEC vaccine ACE527 followed by virulent ETEC H10407 challenge

Article References: Gough, E., Basu, S., Brubaker, J., DeNeraing, B., Sack, D., Bourgeois, A. L., Walker, R., Harro, C. D., & Chakraborty, S. (2026). Influence of gut microbiota on immune responses and protection in volunteers receiving the live attenuated oral ETEC vaccine ACE527 followed by virulent ETEC H10407 challenge. Gut Pathogens. https://doi.org/10.1186/s13099-026-00878-6

Image Credits: AI Generated

DOI: 10.1186/s13099-026-00878-6

Keywords: ETEC, gut microbiota, ACE527 vaccine, H10407 challenge, IgA response, mucosal immunity, 16S rRNA sequencing, diarrhea, vaccine immunogenicity, microbiome diversity, Influence, microbiota

Spleen deficiency with dampness pattern diarrhea is a diagnosis rooted in traditional Chinese medicine that overlaps substantially with chronic functional diarrhea and diarrhea-predominant irritable bowel syndrome as understood in Western medicine. In traditional medical theory, the condition arises when the digestive system loses its capacity to transform and transport fluids, producing loose stools, fatigue, poor appetite, and abdominal discomfort. Clinically, Weiling Decoction has been used for such patients for years, but the pharmacological basis of its effects has remained poorly characterized. The new study set out to close that gap by combining modern analytical chemistry with high-throughput genomic sequencing.

The research team, led by corresponding author Ying Cai, first established a mouse model of the syndrome by exposing Kunming mice to high-humidity environments and administering oral lard, a combination designed to replicate the cold, damp conditions and dietary factors associated with the disorder. The team then turned to an ultra-high-performance liquid chromatography tandem mass spectrometry workflow, known as UHPLC-MS/MS, to chemically fingerprint Weiling Decoction. This analysis identified twenty primary bioactive compounds in the formula, including vicenin-1, inosine, and chlorogenic acid, each of which has documented anti-inflammatory or metabolic activity in previous literature.

With the chemical profile in hand, the researchers evaluated how the formula performed therapeutically. Histological examinations of small intestinal tissue revealed that treated mice showed a marked restoration of villus length, the finger-like projections that absorb nutrients in the gut and are often damaged or shortened in diarrheal disease. Treated animals also exhibited a higher density of goblet cells per unit area, specialized cells that produce the protective mucus layer lining the intestinal wall. These structural changes suggest that the decoction does more than suppress symptoms; it appears to actively support the repair and maintenance of the intestinal mucosal barrier.

Biochemical assays reinforced the picture of a remedy that rebalances disrupted signaling networks. Serum levels of cyclic adenosine monophosphate, or cAMP, a molecule central to regulating fluid secretion in the gut, rose in treated mice alongside increases in D-xylose, a marker of intestinal absorptive capacity, and gastrin, a hormone that stimulates digestive function. Meanwhile, levels of cyclic guanosine monophosphate, or cGMP, and vasoactive intestinal peptide, or VIP, both of which promote intestinal secretion and can exacerbate diarrhea when overproduced, were reduced. Together these shifts indicate that the formula helps recalibrate what the researchers describe as the gastrointestinal-water-energy regulatory network, a coordinated system of hormonal and second-messenger signals that governs how the gut manages fluids and energy.

The heart of the study lies in its microbiome analysis. Using 16S rRNA gene sequencing, the researchers surveyed the bacterial populations in the small intestinal contents of the mice and found that treatment with Weiling Decoction significantly increased the relative abundance of three bacterial genera: Limosilactobacillus, Dwaynesavagella, and Paramuribaculum. Limosilactobacillus, a genus that includes well-known probiotic species, has been repeatedly linked to gut barrier protection and anti-inflammatory effects. Shifts in these bacterial populations suggest the decoction works in part by nurturing a microbial community better equipped to maintain intestinal homeostasis.

To probe what these microbial changes might mean functionally, the team applied PICRUSt2, a computational tool that predicts the metabolic capabilities of microbial communities based on their genetic profiles. The analysis pointed to secondary bile acid biosynthesis as a potentially critical mechanistic pathway. Secondary bile acids are produced when gut bacteria chemically modify the bile acids released by the liver, and a growing body of research links these microbial metabolites to intestinal immune regulation, epithelial barrier integrity, and metabolic signaling. The finding is consistent with recent work showing that gut symbionts can alleviate metabolic and inflammatory disease through secondary bile acid pathways, and it places Weiling Decoction’s effects within a rapidly expanding framework of microbiome-mediated pharmacology.

The study’s authors are careful to frame their findings as correlative rather than definitively causal, noting that the results suggest a microbiota-associated pharmacological mechanism rather than proving one outright. Direct evidence that transplanting the altered microbiota reproduces the therapeutic effect, for instance through fecal microbiota transplantation experiments, remains a logical next step. Nonetheless, the convergence of histological repair, biochemical normalization, and microbial restructuring in the same animals provides a coherent and testable model for how the formula may exert its clinical effects.

From a broader perspective, the research speaks to a growing scientific interest in what traditional Chinese medicine practitioners call syndrome-matched treatment, the idea that therapies should be tailored not just to a disease label but to the specific pattern of dysfunction an individual patient exhibits. By characterizing both the chemical constituents of Weiling Decoction and the molecular and microbial consequences of its administration, the study provides what the authors describe as a biotechnological basis for such syndrome-targeted approaches to gastrointestinal disorders. The data underlying the microbiome analysis have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1346698, allowing other researchers to scrutinize and extend the findings.

Chronic diarrhea remains a substantial global health burden, and conventional treatments often manage symptoms without addressing the underlying disruptions in gut ecology and signaling that drive recurrence. If the mechanisms identified in this mouse model hold up in further studies, including controlled human trials, Weiling Decoction could offer a template for developing microbiome-directed therapies that restore gastrointestinal function by working with, rather than against, the body’s resident microbial communities. For now, the study stands as a compelling example of how modern analytical tools, from mass spectrometry to gene sequencing, can illuminate the biological logic hidden within traditional remedies and potentially translate ancient clinical wisdom into the pharmacological language of the twenty-first century.

Subject of Research: Weiling Decoction alleviates spleen deficiency with dampness pattern diarrhea through modulation of intestinal microbiota

Article Title: Integrated UHPLC–MS/MS and 16S rRNA sequencing reveals that Weiling Decoction alleviates spleen deficiency with dampness pattern diarrhea by modulating intestinal microbiota

Article References: Yu, D., Long, Q., Tian, Q., Zhang, X., Li, D., Tan, Z., & Cai, Y. (2026). Integrated UHPLC–MS/MS and 16S rRNA sequencing reveals that Weiling Decoction alleviates spleen deficiency with dampness pattern diarrhea by modulating intestinal microbiota. 3 Biotech, 16(10), Article 421. https://doi.org/10.1007/s13205-026-05052-y

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05052-y

Keywords: Weiling Decoction, traditional Chinese medicine, intestinal microbiota, diarrhea, 16S rRNA sequencing, UHPLC-MS/MS, spleen deficiency with dampness pattern, secondary bile acids, gut barrier, PICRUSt2, Limosilactobacillus, gastrointestinal health