Microbiome research has identified many highly valuable associations between microbial communities and factors such as diet, antibiotics use, lifestyle, inflammation, infection and chronic disease. However, many findings remain difficult to translate into clinical practice because they are only association-based and vulnerable to confounding. Many studies also often lack reproducibility. Comparisons between "dysbiotic" and "healthy" microbiome states can be misleading because microbiome profiles are shaped by factors such as diet, medication, geography, host genetics, demographics, disease severity and reverse causation. Moreover, there remains no universally accepted definition or consensus regarding what constitutes a ''healthy'' microbiota. What in one individual may be considered a healthy mix of species may not necessarily serve another individual whose diet and lifestyle is completely different. The microbiome research must move beyond descriptive profiling toward clinically actionable and mechanistically informed study designs. The key barriers include small cohort sizes, inconsistent sample collection and treatment, differences in sequencing methods, constantly evolving bioinformatic pipelines, limited availability of raw data, and differences between OTU- and ASV-based analyses. To support a large-scale population studies and meta-analysis a transparent reporting along with shared analytical workflows are essential for progress in this field. Microbiome signatures should not only be reproducible, biologically plausible and modifiable but also linked to the treatment decisions or improved outcomes. This requires longitudinal modelling, external validation, mechanistic studies, and eventually interventional or pragmatic trials. The need for standardisation and reproducibility in microbiome research is widely recognised. This paper builds on that foundation by proposing an actionability-focused framework for designing translational microbiome studies. Using Type 2 Diabetes treatment as a worked example demonstrates that microbiome features should be evaluated not only for their statistical associations, but also for the additional predictive value they provide beyond established clinical variables.

Selective antimicrobial susceptibility supports antimicrobial stewardship strategy but may have limitations in high-resistance prevalence settings. We report a community-acquired infection of necrotic metastatic inguinal lymphadenopathy caused by two mcr-producing Escherichia coli isolates, including one blaOXA-48 producer. Initial selective susceptibility testing delayed recognition of clinically relevant resistance. This case highlights the risk of silent community dissemination of high-priority resistance determinants. It underscores the need for context-adapted susceptibility reporting to balance antimicrobial stewardship and resistance surveillance.

Pathogenic Shiga toxin-producing Escherichia coli (STEC) are a major public health concern and a key issue for the agri-food sector. However, their isolation remains challenging because STEC lack distinctive biochemical characteristics that clearly differentiate them from other Escherichia coli. This study evaluated the performance of nine commonly used agar media for STEC isolation using three types of samples: pure cultures, experimentally spiked food matrices, and naturally contaminated foods. Two isolation approaches were compared: direct streaking and immuno-separation followed by plating. In pure cultures, five media (TBX, SMAC, Rainbow®AgarO157, CHROMID®EHEC Agar, and IDEA™ STEC with or without cefixime-tellurite) achieved complete recovery, whereas CT-CHROMID®EHEC, CT-SMAC and CHROMagarTMSTEC agar inhibited the growth of several strains, particularly those sensitive to tellurite. In food matrices, immunomagnetic separation resulted in high and comparable detection rates across media (80-95%; n = 16/20-19/20). In contrast, direct streaking showed highly variable recovery depending on the medium (29-100%; n = 7/20-20/20). These results demonstrate that combining selective and non-selective media improves STEC detection. Immuno separation enhances recovery of key serogroups, while IDEA STEC enables additional differentiation through halo formation. The observed variability in toxin production suggests that environmental and physiological factors influence STEC detection.

The efficacy of antimicrobial therapy against Shiga toxin-producing Escherichia coli (STEC) infections has been a matter of global debate. In Japan, fosfomycin is often prescribed for pediatric patients to prevent life-threatening sequelae. Recently, E. coli strains harboring plasmid-mediated fosfomycin-resistant genes (fos genes) have been disseminated worldwide, yet the prevalence of these genes among STEC remains unclear. This study aimed to determine the prevalence of fosfomycin resistance among STEC strains isolated from humans, as well as to analyze the molecular characteristics of a conjugative plasmid carrying a fos gene in a STEC strain. Two fosfomycin-resistant STEC O26:H11 strains were identified among 206 STECs by the disk diffusion method. The primary fos gene, fosA3, was identified on an IncF2:A-:B- plasmid in one strain. This plasmid could be transferred to recipients, and the frequency of transfer was higher for STEC O26 recipients compared to STEC O157 recipients. Sequence-based comparative analysis of the plasmid suggested horizontal transfer of fosA3 gene among diverse bacterial strains, with insertion sequences playing a pivotal role in its mobilization. These findings suggest the possibility of fosA3 gene disseminating among diverse STEC strains and emphasize the importance of monitoring fosfomycin resistance and characterizing resistance plasmids in STEC strains.

White grub (Holotrichia serrata F.) is a destructive soil-dwelling pest causing severe yield losses in several agricultural and horticultural crops. Owing to its subterranean habit, chemical control is often ineffective, necessitating sustainable biological alternatives. This study investigated the distribution of coleopteran-active cry genes among indigenous Bacillus thuringiensis strains and evaluated their insecticidal activity against H. serrata. A total of 100 indigenous B. thuringiensis strains isolated from the Western Ghats of India were screened using gene-specific PCR for 12 coleopteran-active cry genes. The most prevalent genes were cry7 (66%), cry26 (65%), cry1b (64%), and cry34 (63%), whereas cry8 and cry9 occurred in fewer than 10% of the isolates. Preliminary bioassays at 380 µg mL[-1] identified 20 strains causing > 50% larval mortality. Concentration-dependent bioassays showed that strain NBAIR Bt68 was the most virulent, causing 73% mortality with an LC50 of 149.95 µg mL[-1]. Highly toxic strains consistently possessed the cry7-cry26-cry34-cry35 gene combination, whereas the total number of cry genes did not correlate with high insecticidal activity. Molecular profiling combined with bioassay evaluation identified indigenous B. thuringiensis strains with promising biocontrol potential and provides insights into the association between cry gene combinations and insecticidal activity against H. serrata.

Heteroresistance, where a small subpopulation of resistant cells coexists within an otherwise susceptible population, plays a critical role in bacterial survival during antibiotic exposure. Yet, its influence on the invasion success of resistant strains remains poorly understood. In this study, we investigate the influence of bacterial heteroresistance, growth, and antibiotic selection on the outcome of an invasion experiment. We quantified the invasion dynamics of a colistin-resistant Pseudomonas aeruginosa strain across a colistin concentration gradient during co-culture with colistin-susceptible clinical P. aeruginosa isolates with varying levels of heteroresistance. The observed variation in heteroresistance and growth allowed investigation of the impact of these factors on invasion dynamics. Our results show that in antibiotic-free conditions, faster-growing isolates competitively excluded the colistin resistant invader, while isolates with high levels of heteroresistance failed to do so. This competitive landscape shifted with increasing colistin concentration, giving the invader a fitness advantage that peaked around 2-4 × MIC of the clinical isolate. The invasion dynamics was further influenced by the associated growth characteristics in the clinical isolates. These findings reveal how both heteroresistance and competitive exclusion shapes invasion dynamics. Understanding these interactions is critical for redesigning treatment strategies that minimize ecological opportunities for resistant strains to establish and expand.

Cylindrospermopsin (CYN) is a cyanotoxin produced by cyanobacterial blooms found in brackish and fresh water. CYN is implicated in toxicity to fish, cattle, and humans, by contaminating water, affecting millions of people worldwide. The limitations of current detection methods of CYN, like chromatography and ELISA, are that they cannot differentiate between the deactivated harmless form and the active cytotoxic harmful form of toxin. The only current test to detect biologically active CYN is a mouse bioassay. We show here that CYN inhibits, in a dose-dependent manner, cell metabolic activities and transduced viral replication and protein expression. We used these findings to develop novel in vitro activity assays to identify, quantify, and differentiate the harmful active CYN from the harmless inactive form. These new in vitro cell-based activity assays have similar sensitivities to that of the currently used in vivo LD50 mouse bioassay. Utilizing these in vitro methods, we also demonstrate that CYN is heat-resistant but can be inactivated by pretreatment with H2O2 at a neutral pH after exposure to direct sunlight. Our results suggest that these in vitro assays can be used as an alternative to animal use, to evaluate the presence of biologically active cytotoxic CYN, to ensure water safety.

Fish gut microbiome research primarily focuses on bacterial communities, while intestinal fungi are often treated as low-abundance background signals or environmental contaminants. Here, we argue that this asymmetry is no longer justified. Evidence from fish systems demonstrates recurrent gut-associated fungi, context-dependent assembly shaped by the host and environment, and multiple routes by which fungi influence host outcomes despite low read fractions. These include extracellular enzymatic activity, immune modulation, and cross-kingdom ecological interactions. Importantly, low fungal read fractions do not indicate low abundance or activity, as cross-kingdom sequencing comparisons are complicated by technical factors, including differential DNA extraction and host DNA contamination. Integrating fungi into conceptual models of the fish gut microbiome requires moving beyond abundance-based interpretations toward a framework grounded in ecological filtering, functional specialization, and redundancy.

Host-associated microbiomes play vital roles in species' ecology and evolution, yet little is known about their shift during the transition from aquatic to terrestrial habitats. We characterize the skin microbiomes of three combtooth blenny species (Blenniella paula, Praealticus labrovittatus, Alticus arnoldorum) that occupy distinct positions along the intertidal gradient-from fully subtidal to intertidal and supratidal environments. Using 16S rRNA sequencing, we compared skin-associated bacterial communities with those in seawater and substrate biofilms. The divergence between skin and substrate microbiomes parallels fishes' distribution along progressively higher intertidal zones. The supratidal A. arnoldorum had the most divergent microbiome, characterized by higher Gammaproteobacteria abundance and enrichment of epiphytic and mucus-associated taxa. Fish skins were enriched in 27 microbial orders that included genera commonly associated with fish (e.g. Vibrio, Alteromonas, Cetobacterium) but others rarely reported (e.g. Rubritalea, Granulosicoccus). Together, our findings suggest that variation in fish skin microbiomes parallels the ecological transition from subtidal (aquatic) to supratidal (terrestrial) habitats, although host and habitat effects could not be disentangled. We hypothesize that microbial symbionts may contribute to adaptations enabling amphibious lifestyles. This is the first study on fish microbiome variation across the intertidal gradient and offers a framework for predicting microbiome responses to environmental change.

Microbiology students often struggle with the extensive and specialized terminology of the discipline, frequently relying on rote memorization rather than understanding word structure, which limits long-term retention. This study investigated the integration of morphological and etymological analysis into microbiology courses as a tool to support vocabulary learning. Surveys conducted at the end of the semester over three academic years revealed that students generally accepted the approach, reported an improved ability to memorize and understand terms, and indicated that they would use it in the future to decode unfamiliar scientific words. These results suggest that morpheme-based instruction represents a practical and promising strategy for enhancing comprehension and the retention of microbiological terminology.

The evolution of phototrophy and carbon fixation in the phylum Chloroflexota remains incompletely understood, largely due to the scarcity of cultured mesophilic representatives. Here, we report the isolation and genomic characterization of a novel filamentous anoxygenic phototrophic bacterium from a microbial mat in the mesothermal Lake Umkhey (Southern Siberia). Although the original culture was lost, its genome was recovered via metagenome-assembled genome reconstruction from environmental samples, enabling a detailed phylogenomic and metabolic analysis. The genome of umkhey_bin13 (proposed as 'Candidatus Viridifilum namsaraevi' gen. nov., sp. nov.) encodes a complete 3-hydroxypropionate bicycle for autotrophic CO₂ fixation, a type II sulfide:quinone oxidoreductase, but lacks nitrogenase and chemolithotrophic markers. Comparative genomics across 18 Chloroflexota genomes reveals that this new lineage occupies an intermediate phylogenetic position, with distinct gene content for sulfur, nitrogen, and hydrogen metabolism, and highlights a complex evolutionary history within the group. Our findings provide new insights into the evolutionary transitions of photosynthetic and carbon-fixation pathways, and underscore the value of integrating cultivation-independent genomics with classical microbiology. The proposed novel genus and species contribute to a revised framework of Chloroflexota evolution and offer a genomic reference for studying metabolic adaptation in alkaline, sulfide-influenced microbial mats.

Ehrlichia ruminantium, the causative agent of heartwater disease, is an obligate intracellular bacterium that poses significant economic threats to livestock production in endemic regions. Current research models present substantial ethical, logistical, and economic constraints, particularly for studying host-pathogen interactions within arthropod vectors. Here, we establish Galleria mellonella larvae as a tractable invertebrate infection model for Ehrlichia ruminantium, enabling experimental investigation of pathogen persistence and host-pathogen interactions in an arthropod system. Following infection, G. mellonella proved susceptible to E. ruminantium with moderate mortality and remarkable bacterial persistence. Using rhodamine-labeled bacteria and fluorescence microscopy, we tracked bacterial dissemination from injection sites to systemic distribution in characteristic segmental patterns throughout the larval body. Rhodamine-labeled bacteria were consistently associated with hemocytes, the primary immune cells of G. mellonella, although widefield imaging cannot establish intracellular localization. Quantitative PCR revealed stable bacterial loads over the study period, and bacteria recovered from infected larvae remained viable and infectious for endothelial cells. These findings show that E. ruminantium persists in G. mellonella for at least two weeks without inducing a detectable melanization response. The fluorescence adopted a reproducible segmental distribution whose biological basis remains to be determined. This model offers significant advantages: ethical acceptability, cost-effectiveness, experimental tractability, and compatibility with high-throughput screening approaches. The G. mellonella system represents a valuable complement to existing mammalian models and provides a unique platform for investigating arthropod-specific aspects of E. ruminantium biology, screening antimicrobial compounds, and testing hypotheses of immune evasion that may inform strategies for heartwater disease control.

Alexandrium catenella (Group I) causes paralytic shellfish poisoning. Nitrogen is a critical component for both growth and toxin production in A. catenella, and recent studies have indicated that A. catenella uses ammonium as a nitrogen source. However, the molecular mechanism underlying the transportation of ammonium in A. catenella has not been identified. In this study, we attempted to isolate and characterize the ammonium transporter (AMT) in A. catenella. cDNA cloning produced a number of AMT clones. To investigate the ammonium transport capability of A. catenella AMT (AlcAMT), we selected three representative cDNAs and the clones were inserted into expression vectors. These vectors were then transferred into an amt-deficient Schizosaccharomyces pombe transgenic yeast strain, and the resulting transformants were grown on a medium containing NH4Cl as the only nitrogen source. Following transformation, only yeast transformants carrying one of the three AMTs were able to grow on the medium, confirming its ability to transport ammonium. Sequence analysis of the three AMT variants revealed that the successful variant included a K residue at sequence position 450, while the other variants contained a T residue at the same position. Mutation analysis confirmed that K450 is important for the function of AlcAMT.

Edwardsiella anguillarum is a bacterial pathogen affecting diverse aquaculture species worldwide, including red seabream, Japanese eel, and tilapia. In Japan, strains show a phenotypic dichotomy, where red seabream-derived strains are non-motile and ornithine decarboxylase (ODC)-negative, whereas eel-derived strains retain both traits. However, the evolutionary processes underlying lineage diversification within this species remain poorly understood. We performed a global comparative genomic analysis of 24 E. anguillarum strains, including seven newly sequenced genomes from Japan. Despite high ANI values (>99.6%), SNP analysis revealed lineage-level divergence, with 6500-7500 SNPs separating red seabream-associated and eel-associated lineages. Comparative analysis identified a conserved chromosomal hotspot adjacent to the tRNA-Thr gene with lineage-specific structural variation. The Japanese red seabream-associated lineage possessed a compact ∼10-kb region containing a Type I-E CRISPR/Cas system, whereas eel-associated and tilapia-derived strains carried expanded genomic islands (∼48-52 kb) enriched in DNA repair and mobile genetic element functions. These configurations were associated with phylogeographic lineages. Genes associated with motility and ODC activity were conserved across lineages, with no evidence of gene loss or major structural disruption. Phylogenetic analysis of the island-associated tyrosine-type recombinase/integrase (YR) was broadly consistent with the core genome. A shared frameshift mutation in eel- and tilapia-derived strains was consistent with a shared pseudogenization event. Overall, our findings indicate that diversification in E. anguillarum is associated with localized structural variation at a conserved tRNA-Thr-associated chromosomal hotspot within an otherwise highly conserved core genome.

Burkholderia semiarida, a plant-pathogenic species within the Burkholderia cepacia complex (Bcc), has emerged as a worrying opportunistic pathogen causing serious respiratory infections in patients with cystic fibrosis (CF). In this study, strain SYBS718 was isolated from the blood and sputum of an immunocompromised patient. Conventional MALDI-TOF mass spectrometry and 16S rRNA sequencing misidentified this isolate as Burkholderia cenocepacia, whereas whole-genome sequencing enabled accurate species confirmation. Genomic analysis predicted a total of 569 virulence factors, 11 antibiotic resistance genes, and 1112 type III secretion system effector proteins related genes through the core databases of VFDB, CARD, and TS33. The virulence factors mainly include type VI secretion system (T6SS), type III secretion system (T3SS), iron uptake system, flagella, and type IV pili (TFP), which are associated with bacterial colonization, invasion, and pathogenicity. And the antibiotic resistance genes are primarily associated with fluoroquinolone, tetracycline, and aminoglycoside antibiotics. This study is the first to perform comprehensive genomic characterization of a clinical B. semiarida isolate. Our findings reveal the limitations of routine Bcc identification, and provide fundamental genomic data for further clinical and mechanistic research on this emerging opportunistic pathogen.