To appreciate why ERMA matters, it helps to understand what epicPCR actually does. Emulsion, Paired Isolation and Concatenation PCR, to give the technique its full name, is a single-cell method that physically links functional genes to taxonomic marker sequences from the same microorganism. Individual cells are encapsulated in emulsion droplets, their genomes are fragmented, and a fusion PCR step concatenates a functional gene of interest, for example an antibiotic resistance gene, with the 16S rDNA marker that identifies which organism carried it. Sequencing the resulting fusion products then allows researchers to answer a question that conventional metagenomics struggles with: which microbes in a complex community actually carry which functional genes. This capability has made epicPCR increasingly important in microbial ecology and, above all, in antimicrobial resistance research, where surveillance of wastewater and other environmental reservoirs depends on knowing not just which resistance genes are present but which organisms harbor them.

The catch has been the analysis. Until now, most laboratories processed epicPCR sequencing output using custom, often unpublished scripts, tailored to individual studies and rarely shared in reusable form. That fragmentation made cross-study benchmarking difficult, reuse nearly impossible, and reproducibility a matter of goodwill rather than infrastructure. Two laboratories analyzing the same dataset could, and routinely did, arrive at different taxonomic assignments and different resistance gene inventories simply because their filtering rules and database choices differed. In a field where wastewater-based epidemiology is being actively developed for public health decision-making, such inconsistency is more than an inconvenience; it undermines confidence in the underlying measurements.

ERMA tackles the problem head-on with a fully automated, standardized workflow built on Snakemake, a widely used workflow management system that makes computational pipelines transparent, restartable, and scalable. The pipeline requires minimal user input and supports both Illumina short-read and Oxford Nanopore Technologies long-read sequencing data, a notable flexibility given that epicPCR studies have historically been split between these platforms. From raw sequencing data, ERMA carries the analyst through quality control, reference database preparation, dual similarity searches against separate taxonomic and functional databases, integration of the two result streams, filtering, and finally visualization of the linked taxonomic-functional pairs. Every step is documented and executed identically across runs, which is precisely what the field has lacked.

The modular design is a deliberate architectural choice with consequences beyond antimicrobial resistance. Because database preparation is modular and the pipeline can incorporate specific UniRef queries, researchers can retarget ERMA to functional genes other than resistance determinants. A team interested in plastic-degradation genes, virulence factors, or metabolic pathways can in principle swap in the appropriate reference sets without rewriting the pipeline. This adaptability positions ERMA as a general-purpose epicPCR analysis engine rather than a single-purpose resistome tool, and the authors explicitly frame it as suitable for a broad range of research questions across microbial ecology.

Validation was carried out on two fronts. First, the team benchmarked ERMA against five publicly available epicPCR datasets spanning diverse sample sources and sequencing depths, from environmental samples to clinical material. Across these heterogeneous datasets, ERMA achieved mean concordance rates of approximately 70 to 83 percent with the taxonomic results reported in the original publications. Given that the original studies each used bespoke analysis approaches, this level of agreement suggests that a single standardized workflow can recover the core biological signal of epicPCR experiments while making the processing steps explicit and comparable. The residual differences between ERMA’s output and the published results are themselves informative, since they localize where analytical choices, rather than biology, drive divergence between studies.

The second validation was more direct. The researchers spiked a defined five-member mock community into clinical wastewater, a matrix notorious for its chemical complexity and microbial diversity, and ran it through the pipeline. ERMA recovered all five mock genera in full, a result that speaks to the pipeline’s sensitivity even in a realistic, high-background sample. As an independent cross-check, the same wastewater sample was analyzed by conventional 16S rDNA gene sequencing, and 85 percent of the genera detected by ERMA were also detected by that orthogonal method. For a technique that links function to identity at the single-cell level, agreement with an established amplicon-based approach at the genus level provides meaningful external validation that the fusion products are being correctly parsed and assigned.

One of the study’s more sobering findings came from the attrition analysis, which tracked how sequencing reads are lost at each filtering stage. The comparison revealed inconsistent filtering patterns across the five public datasets, reflecting dataset-specific differences in quality. In other words, the amount of usable information extracted from an epicPCR experiment depends heavily on the quality of the starting material and the sequencing run, and until now those losses were invisible, buried inside custom scripts. By making attrition explicit and standardized, ERMA gives researchers a diagnostic instrument: laboratories can now see where their data degrade and adjust library preparation or sequencing depth accordingly, rather than discovering problems only after publication.

The authors are candid about the field’s remaining limitations. Large-scale reference datasets for epicPCR remain scarce, which constrains how thoroughly any analysis pipeline, however well built, can be benchmarked. There is no vast, curated corpus of epicPCR data comparable to what exists for shotgun metagenomics, and building one will require the kind of shared tooling that ERMA now provides. Still, the combination of concordance with published results, full recovery of a mock community in clinical wastewater, and strong agreement with independent 16S rDNA analysis demonstrates strong and interpretable performance across genuinely heterogeneous inputs, which is arguably the hardest test a general pipeline can face.

The significance of ERMA extends beyond its immediate user base. Wastewater-based surveillance of antimicrobial resistance has moved from academic curiosity to policy relevance, with public health agencies worldwide investing in monitoring programs, and the WBEready consortium, funded by the German Federal Ministry for Health, supported this work as part of that broader effort. Surveillance only works if measurements from different laboratories and different time points can be compared, and that comparability is exactly what standardized analysis delivers. By providing a transparent, modular, and reproducible workflow as open-source software, ERMA establishes a foundation for methodological standardization and future benchmarking efforts in epicPCR, and as reference datasets grow, the pipeline’s performance should improve in step. For a technique that can reveal which bacteria in a wastewater stream carry which resistance genes, that kind of computational discipline may prove as important as any wet-lab innovation.

Subject of Research: Standardized computational analysis of epicPCR data linking functional genes to microbial taxonomy

Article Title: ERMA: a harmonizing epicPCR data analysis tool

Article References: Dörr, A., Dekić Rozman, S., Duncker, L. V., Kehrmann, J., Buer, J., Virta, M., Meyer, F., & Kraiselburd, I. (2026). ERMA: a harmonizing epicPCR data analysis tool. BMC Genomics. https://doi.org/10.1186/s12864-026-13418-y

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13418-y

Keywords: epicPCR, ERMA, Snakemake, antimicrobial resistance, wastewater surveillance, microbial ecology, bioinformatics pipeline, Illumina sequencing, Oxford Nanopore, 16S rDNA, reproducibility, resistome

The study focused on Niğde province, a region of central Türkiye where tortoises roam freely and their parasites follow. The research team, led by Bilge Karatepe and Mustafa Karatepe of the Department of Biotechnology, together with Fatma Mutlu and Zeynep Ergüleş, examined 88 spur-thighed tortoises, Testudo graeca, a species whose conservation status makes every detail of its parasite burden relevant. From these animals they collected 184 adult ticks, 167 males and just 17 females, preserving each specimen in sealed bottles of 70 percent alcohol before carrying them to the laboratory for identification under a stereo microscope.

Every single tick turned out to be Hyalomma aegyptium, confirming the reputation of this species as the quintessential tortoise tick. The infestation pattern was itself informative. Ticks were found on 62 of the 88 tortoises examined, an infestation rate of 70.4 percent. When the researchers broke the data down by month, they discovered that infestation rates varied significantly across the season, a statistically robust pattern with a p value below 0.05. The sex distribution of ticks on infested hosts, however, showed no significant variation, suggesting that male and female ticks exploit their reptile hosts in broadly similar ways even though males vastly outnumbered females in the sample.

With the ticks identified and counted, the team turned to the molecular heart of the study. Rather than testing each tick individually, they organized the 184 specimens into 68 pools, with separate pools for male and female ticks collected from each tortoise. This pooling strategy is a standard and efficient approach in tick microbiology, allowing researchers to screen large numbers of arthropods for rare infections without running hundreds of separate reactions. DNA was extracted from each pool and subjected to polymerase chain reaction, or PCR, using primers specifically designed to amplify marker genes from two bacterial genera of great interest to tick biologists.

The first target was Wolbachia, arguably the most widespread endosymbiotic bacterium on Earth. Wolbachia infects an enormous range of arthropods, manipulating the reproduction of its hosts in ways that can skew sex ratios, kill males, or enable reproductive parasitism. Because of these dramatic effects, Wolbachia has become a darling of both evolutionary biology and pest control, engineered into mosquito populations to block the transmission of dengue and other viruses. The researchers targeted the wsp gene, a surface protein gene of about 610 base pairs that serves as a reliable molecular fingerprint for the bacterium. The outcome was unambiguous: not one of the 68 tick pools produced a Wolbachia amplification product. Only the positive control, supplied by Serap Aksoy of the Yale School of Public Health, yielded the expected 610 base pair band, confirming that the assay worked perfectly and that the negative results were real biology rather than technical failure.

The second target told a different story. The team used primers directed at the 16S ribosomal RNA gene of Spiroplasma, a roughly 1000 base pair region that serves as the gold standard barcode for identifying bacteria. Spiroplasma is a fascinating genus of spiral-shaped, wall-less bacteria in the class Mollicutes, relatives of Mycoplasma. These microbes are best known from insects and plants, where they can act as pathogens, mutualists, or reproductive manipulators. In some insect species, Spiroplasma strains are male killers, selectively destroying male embryos to favor their own transmission through infected females, a phenomenon documented in organisms such as the small brown planthopper. In ticks, Spiroplasma has been reported sporadically, and its ecological role remains poorly understood.

When the researchers ran their Spiroplasma assays, four of the 68 pools, or 5.8 percent, came back positive for Spiroplasma DNA. Every one of the positive pools originated from male ticks, an intriguing observation given that the sample was overwhelmingly male to begin with. The amplified 16S rDNA fragments were analyzed with molecular evolutionary software, allowing the team to characterize the endosymbionts genetically and confirm their identity as Spiroplasma species. This detection represents the first molecular evidence of Spiroplasma in Hyalomma aegyptium ticks in Central Anatolia, a region where tick microbiology has been studied far less intensively than in coastal or western parts of the country.

The absence of Wolbachia is arguably as interesting as the presence of Spiroplasma. The authors note that their null result is consistent with several recent studies reporting low or absent Wolbachia prevalence in ixodid, or hard, ticks. This pattern contrasts sharply with the situation in soft ticks and in many insects, where Wolbachia is common. The reasons remain debated. Some researchers suggest that the physiology of hard ticks, including their long feeding intervals and unique reproductive biology, may simply be inhospitable to Wolbachia colonization. Others point to the possibility that competitive exclusion by other endosymbionts, such as Coxiella-like or Francisella-like bacteria that frequently dominate tick microbiomes, keeps Wolbachia out. Whatever the mechanism, the growing consensus is that hard ticks are not the Wolbachia reservoirs that earlier assumptions based on insect data might have predicted.

The tortoise tick itself deserves attention as a microbial vehicle. Hyalomma aegyptium has been implicated as a long-term carrier of Coxiella burnetii, the agent of Q fever, and previous work in Türkiye and neighboring countries has detected Borrelia turcica, Rickettsia species, Ehrlichia, and the endosymbiont Candidatus Midichloria mitochondrii in these ticks. Because tortoise ticks feed intensively on reptiles and occasionally attach to other animals, understanding their endosymbiont community helps distinguish true pathogens from harmless or even beneficial residents. Endosymbionts can also shape vector competence, the ability of a tick to acquire, maintain, and transmit disease agents, so mapping which microbes are present is a prerequisite for assessing public health risk. The Niğde survey contributes to a broader effort, spanning studies from Algeria and Qatar to Israel and the Balkans, to catalog the microorganisms associated with this single tick species across its vast range.

For the tortoises themselves, the findings carry conservation implications. Testudo graeca is under pressure across its range from habitat loss and collection, and heavy tick burdens can impose physiological costs on already vulnerable populations. Epidemiological studies of tick infestation in spur-thighed tortoises have begun to unravel the demographic consequences of parasitism, and knowing which microbes circulate in the ticks adds a layer of understanding about disease dynamics in these reptile communities. The Niğde study, supported by the Scientific Research Projects Coordination Unit of Niğde Ömer Halisdemir University, also demonstrates the value of regional molecular surveillance. A single season of fieldwork in one Anatolian province produced a novel regional record for Spiroplasma and a robust negative result for Wolbachia, both of which refine the emerging global picture of tick microbiomes. As climate trends continue to alter the distribution and activity of ticks worldwide, baseline data like these will become ever more valuable for predicting where tick-borne agents may emerge next, and for separating the genuine threats from the hidden symbionts that simply ride along.

Subject of Research: Molecular detection and characterization of Spiroplasma and Wolbachia endosymbionts in Hyalomma aegyptium ticks infesting spur-thighed tortoises in Central Anatolia

Article Title: Molecular Characterization of Spiroplasma Endosymbionts in Ticks on Tortoises in Niğde Province, Türkiye

Article References: Karatepe, B., Karatepe, M., Mutlu, F., & Ergüleş, Z. (2026). Molecular Characterization of Spiroplasma Endosymbionts in Ticks on Tortoises in Niğde Province, Türkiye. Acta Parasitologica, 71(5), Article 224. https://doi.org/10.1007/s11686-026-01410-1

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01410-1

Keywords: Hyalomma aegyptium, Spiroplasma, Wolbachia, tick endosymbionts, Testudo graeca, tortoise ticks, Niğde, Türkiye, PCR, 16S rDNA, Central Anatolia, tick microbiome