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bioRxiv2024

Gellan gum-based nutrient medium increases the diversity of cultivated groups of soil microorganisms

Soil microbial communities contain a huge proportion of microorganisms that cannot be cultured on standard microbiological media and are available for study only by molecular methods such as metagenomics. Among them may be found microorganisms-producers of biologically active substances useful for medicine, biotechnology and agricultural activities of humans. Development of approaches for cultivation of such groups may open the way to the creation of fundamentally new antibiotic, antitumor drugs, phyto- and immunostimulants. We have proposed a solid oligotrophic medium for cultivation of soil microorganisms containing gellan gum, which allows to obtain cultures of more rare microorganisms compared to known media. Representatives of phylum Chloroflexota, Deinococcota, Planctomycetota, Actinomycetota, which were absent on agar media, were found in the metagenome of the community grown on this medium

microbiology↗

bioRxiv2024

Rapid rewiring of an archaeal transcription factor function via flexible cis-trans interactions

For microbial cells, an appropriate response to changing environmental conditions is critical for viability. Transcription regulatory proteins, or transcription factors (TFs) sense environmental signals to change gene expression. However, it remains unclear how TFs and their corresponding gene regulatory networks are selected over evolutionary time scales. The function of TFs and how they evolve are particularly understudied in archaeal organisms. Here we identified, characterized, and compared the function of the RosR transcription factor across three related hypersaline adapted archaeal model species. RosR was previously characterized as a global regulator of gene expression during oxidative stress in the species Halobacterium salinarum (hsRosR). Here we use functional genomics and quantitative phenotyping to demonstrate that, despite strong sequence conservation of RosR across species, its function diverges substantially. Surprisingly, RosR in Haloferax volcanii (hvRosR) and Haloferax mediterranei (hmRosR) regulates genes whose products function in motility and outer membrane structure, leading to significant defects in motility when rosR is deleted. Given weak conservation and degeneration in cis-regulatory sequences recognized by the RosR TF across species, we hypothesize that the RosR regulatory network is readily rewired during evolution across related species of archaea. SIGNIFICANCE STATEMENTO_LIGene regulation enables cells to sense and respond to environmental signals. The mechanisms by which gene regulatory circuits change and adapt over evolutionary time scales remain unclear, especially in understudied domains of life like the archaea. C_LIO_LIHere we demonstrate that the archaeal-specific RosR transcription protein plays fundamentally different roles in environmental response of related archaeal species (oxidative stress protection vs motility). This divergence likely occurred through reduced selectivity of RosR for certain DNA sequence motifs. C_LIO_LIThese findings are surprising given strong sequence and structural conservation of RosR and suggest that transcription regulator function can diverge rapidly through flexible protein-DNA interactions. This mechanism of divergence is shared between eukaryotes and archaea, suggesting ancient origins. C_LI Subject categories: Bioinformatics, microbiology, genetics, gene regulatory networks

microbiology↗

bioRxiv2024

Heat stress impacts the gut microbiome of Atlantic salmon by promoting growth of Vibrionaceae and is associated with extensive cast production

Atlantic salmon (Salmo salar) when exposed to heat stress have reduced voluntary feeding and exhibit changes in digesta consistency. This study was performed to determine what bacterial species occur and the overall bacterial abundance in the gut microbiome in heat stressed Atlantic salmon. For this Atlantic salmon in seawater tanks at 15{degrees}C were fed for 2 to 4 weeks. Tank temperatures were increased to 19{degrees}C until voluntary feeding abated at which point tank temperatures were cooled to 15{degrees}C for 4 weeks. At the end of each temperature phase the fish were stripped of feces and microbiome profiles were determined using 16S rRNA V1-V3 metabarcoding. The tank experiment was repeated three times in successive years. Vibrionaceae comprised most reads after the warm phase completed. The prominent levels of Vibrionaceae were accompanied by a large predominance of cast (sloughed intestinal mucosa) containing fecal samples. qPCR estimated Vibrionaceae cell populations increased 1.9-3.4 log units/g after the warm phase but and slightly decreased by 0.3-1.1 log units/g after the 15{degrees}C recovery phase. The results indicated heat stress induced inappetence corresponds to increased cast production accompanied by predominance of Vibrionaceae. Vibrionaceae colonizing the Atlantic salmon gut should be the focus of studies on the microbiology of thermally induced inappetence and dysbiosis in Atlantic salmon. Key ContributionThe experiments confirm that thermal stress increased cast production in Atlantic salmon. The increase in cast production coincides with increased predominance of Vibrionaceae. Though the growth of Vibrionaceae in the gut of salmon is not associated with acute disease it may represent a dysbiosis effect. The results suggest extended summers in regions where sea surface temperatures are at the tolerance limits require increased husbandry management of farmed fish. In addition, more studies are needed to determine if novel approaches can limit overgrowth of Vibrionaceae.

microbiology↗

bioRxiv2024

The impact of fold-dilution and diluent volume on the estimation of colony forming units in a bacterial culture

Considering the non-uniform distribution of bacteria in a liquid culture, accurate enumeration of bacteria by serial dilution is important yet challenging in microbiological research. The deviation of estimated population size from the actual population size is anticipated in view of the sampling error arising at dilution, which is known to increase at each successive dilution; and the sampling error associated with the sample volume, where it is inversely proportional. In the dilution process of such population following a Poisson distribution, the relation of sampling error with the sequentially diluted suspensions is yet to be empirically demonstrated. Also, as a given-fold dilution can be achieved by passaging of either a higher or a lower sample volume, the sampling error associated in this case with the volume being passaged also remains to be evaluated. In view of that, the current experimental study was undertaken to further understand bacterial distribution and the underlying sampling error in dimensions previously not demonstrated. We demonstrated that sampling error in the enumeration process is independent of the bacterial cell densities of the diluted suspensions, which was on contrary to the theoretical assumptions. Moreover, sampling error is also not dependent on the number of passages in the dilution process. Importantly, we also demonstrated that passaging either a higher or lower sample volumes during serial dilution yielded similar enumeration, which was exceptional with regards to the statistical fluctuations inherent to a Poisson distribution.

microbiology↗

bioRxiv2024

Whole genome Sequencing Reveals Enterobacter hormaechei as a key Bloodstream Pathogen in six tertiary care hospitals in southwestern Nigeria.

Enterobacter spp. are an important cause of healthcare-associated bloodstream infections uncommonly reported in Africa. This study used whole genome sequencing (WGS) to characterise Enterobacter spp. from hospitals in Nigerias antimicrobial resistance (AMR) surveillance system. Blood-culture isolates of Enterobacter from six such tertiary-care hospitals recovered between 2014 and 2020 were re-identified and antimicrobial susceptibility-tested using VITEK2. Illumina technology provided whole genome sequences for genome nomenclature, antimicrobial resistance gene prediction, Single Nucleotide Polymorphism (SNP) phylogeny, and multi-locus sequence typing via publicly available bioinformatics pipelines. Initial biochemical delineation often misclassified Enterobacter, necessitating whole-genome sequencing for accurate classification. Among 98 Enterobacter received, Enterobacter hormaechei subspecies xiangfangensis predominated (43), followed by other E. hormachei subspecies (18), E. cloacae (26), E. roggenkampii (4), E. bugandensis (3), E. kobei (2), E. asburiae (1) and E. cancerogenous (1). Cephalosporins, aminoglycoside, chloramphenicol, macrolide, and carbapenem resistance in E. hormaechei was attributed to known resistance genes. They belonged to clusters III, IV, and VIII based on hsp60 typing and clades A, B, C, and D according to Sutton and Cos nomenclature. These isolates and other Enterobacter species recently reported from Nigeria reveal extensive E. hormaechei diversity, as well as clusters representing potential outbreaks. Enterobacter hormaechei, often misidentified and rarely reported from Nigeria, is this studys most common blood culture isolated Enterobacter spp. Uncovering underappreciated species as important bloodstream pathogens and retrospective detection of likely outbreaks emphasise the value of genomic surveillance in resource-limited settings. DATA SUMMARYAll sequence reads were submitted to the European Nucleotide Archive (ENA) under the project ID PRJEB29739 (https://www.ebi.ac.uk/ena/browser/view/PRJEB29739). Accessions can be found in Table S1. IMPACT STATEMENTAccurate identification of Enterobacter is essential in healthcare settings as misidentification can lead to selecting antimicrobials to the genus is intrinsically resistant resistant before susceptibility testing results are available. Also, misidentification can compromise microbiology support for infection prevention and control. We show that E. hormaechei, which is never reported from clinical laboratories in Nigeria, is frequently misidentified using conventional methods like tube- or strip biochemical testing and VITEK systems. Whole genome sequence data demonstrates that E. hormaechei and E. cloacae are the most common Enterobacter isolated from bloodstream infections in Nigeria. Enhanced identification methods for surveillance play pivotal roles in improving patient care, optimising antibiotic stewardship, and combating the evolving challenges posed by this pathogen. Overall, this study reveals the effectiveness of WGS in correctly identifying this important pathogen.

microbiology↗

bioRxiv2024

Cultivation and molecular characterization of Foley catheterized urine, clean catch urine, and vaginal swabs from pregnant women prior to delivery

The bladder and urine have historically been considered sterile, especially in the context of clinical assessment. Yet, enhanced culture techniques and advances in DNA sequencing surveys have revealed a low microbial biomass microbiota in the bladders of most healthy individuals. Yet very few studies have investigated the urinary microbiota of pregnant women, who are at increased risk of urinary tract infections (UTIs) that can lead to pregnancy complications such as spontaneous preterm birth. To better understand the potential role of a bladder microbiota during pregnancy, we characterized the urinary microbiota of 25 pregnant women (delivering at or beyond 36 weeks gestation) by comparing the bacterial profiles of their paired catheter urine, clean catch urine, and vaginal swabs through both cultivation and molecular microbiological survey methods. For culture, three bacterial taxa were detected in at least 20% of all urine samples (Lactobacillus species, coagulase negative Staphylococcus species, and Ureaplasma urealyticum). All three taxa were detected less frequently in Foley catheter urine than in clean catch urine. Ureaplasma urealyticum was the most frequently recovered bacterium in Foley catheter urine (13/25 women). It was also identified as being more relatively abundant in Foley catheter urine than in the vagina through 16S rRNA gene surveys. Other lower abundance Gram-positive anaerobic cocci (i.e., Finegoldia and Anaerococcus) were also more relatively abundant in Foley catheter urine than clean catch urine or vaginal swabs. However, all sample types had high relative abundances of Lactobacillus and Gardnerella species. Overall, this suggests that, although vaginal microbiota contamination cannot be completely avoided, Foley catheterized urine is effective at characterizing the low abundance bladder microbiota, including Ureaplasma, Finegoldia, and Anaerococcus species. This warrants their further consideration as commensal members of the bladder microbiome during pregnancy.

microbiology↗

bioRxiv2024

Chemoreceptor family in plant-associated bacteria responds preferentially to the plant signal molecule glycerol 3-phosphate

Plant pathogens and plant-associated bacteria contain about twice as many chemoreceptors as the bacterial average, indicating that chemotaxis is particularly important for bacteria-plant interactions. However, information on the corresponding chemoreceptors is limited. In this study, we identified the chemoreceptor PacP from the phytopathogen Pectobacterium atrosepticum, which exclusively recognized C3 phosphorylated compounds at its sCache ligand binding domain, mediating chemoattraction. Using a motif of PacP amino acid residues involved in ligand binding, we identified a chemoreceptor family, termed sCache_PC3, that was specific for C3 phosphorylated compounds. Isothermal titration calorimetry studies revealed that family members preferentially bound glycerol 3-phosphate, a key plant signaling molecule. Additionally, family members recognized glycerol 2-phosphate and glycolysis intermediates glyceraldehyde 3-phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate. This study presents the first evidence of chemoreceptors that bind phosphorylated compounds. We show that the sCache_PC3 family has evolved from an ancestral sCache domain that respond primarily to Krebs cycle intermediates. Members of the sCache_PC3 family were mainly found in bacteria that interact with plants, including many important plant pathogens such as Brenneria, Dickeya, Musicola, Pectobacterium, and Herbaspirillum. Glycerol 3-phosphate is a signal molecule that is excreted by plants in response to stress and infection. Chemotaxis towards this molecule may thus be a means for bacteria to localize stressed plants and move to infection sites. This study lays the groundwork for investigating the functional importance of chemotaxis to phosphorylated C3 compounds in plant-bacteria interactions and virulence. Significance statementThe bacterial lifestyle has shaped the evolution of signal transduction systems, and the number and type of chemoreceptors varies greatly between bacteria occupying various ecological niches. Our understanding of the relationship between lifestyle and chemoreceptor function is limited and the discovery of a chemoreceptor family in plant-associated bacteria that primarily responds to an important plant signal molecule is a significant advancement, allowing for further studies to determine its physiological relevance. The lack of knowledge about signals recognized by bacterial receptors is currently a major challenge in microbiology. This study illustrates the potential of combining experimental ligand screening with computational ligand prediction to identify signals recognized by uncharacterized receptors.

microbiology↗

bioRxiv2025

Fusobacterium nucleatum is enriched in invasive biofilms in colorectal cancer

Fusobacterium nucleatum is an oral bacterium known to colonize colorectal tumors, where it is thought to play an important role in cancer progression. Recent advances in sequencing and phenotyping of F. nucleatum have revealed important differences at the subspecies level, but whether these differences impact the overall tumor ecology, and tumorigenesis itself, remain poorly understood. In this study, we sought to characterize Fusobacteria in the tumor microbiome of a cohort of individuals with CRC through a combination of molecular, spatial, and microbiologic analyses. We assessed for relative abundance of F. nucleatum in tumors compared to paired normal tissue, and correlated abundance with clinical and pathological features. We demonstrate striking enrichment of F. nucleatum and the recently discovered subspecies animalis clade 2 (Fna C2) specifically in colon tumors that have biofilms, highlighting the importance of complex community partnerships in the pathogenesis of this important organism.

microbiology↗

bioRxiv2025

Ten species comprise half of the bacteriology literature, leaving most species unstudied

Microbiology research has historically focused on a few species of model organisms. Our bibliographic analysis finds extreme bias in the distribution of bacteriology research across species, with half of all papers referencing only ten species and 74% of all known species remaining unstudied. Microbiologists will need to broaden their perspective and embrace complexity to develop a complete understanding of the microbial world.

microbiology↗

bioRxiv2025

Optimising synthetic cystic fibrosis sputum media for growth of non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) is an early pathogen isolated from the lungs of children with cystic fibrosis (CF). However, its role in the progression of CF lung infection is poorly understood. Additionally, whether it forms biofilms in the lungs of people with CF is an open question. The development of synthetic cystic fibrosis sputum media has given key insights into the microbiology of later CF pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, through replicating the chemical composition of CF sputum. However, growth of NTHi in these media has not previously been reported. We show that NTHi grows poorly in three variants of synthetic cystic fibrosis sputum media commonly used to induce in vivo -like growth of P. aeruginosa and S. aureus (SCFM1, SCFM2 and SCFM3). The addition of NAD and hemin to SCFM1 and SCFM2 promoted the planktonic growth and biofilm formation of both laboratory and clinical NTHi isolates, and we were able to develop a modified variant of SCFM2 that allows culture of NTHis. We show that NTHi cannot be identified in an established ex-vivo model of CF infection, which uses SCFM and porcine bronchiolar tissue. This may in part be due to the presence of endogenous bacteria on the pig lung tissue which outcompete NTHi, but the lack of selective agar to isolate NTHi from endogenous bacteria, and the fact that NTHi is an exclusively human pathogen, make it hard to conclude that this is the case. Through spiking modified SCFM2 with filter sterilized lung homogenate, biofilm growth of clinical NTHi isolates was enhanced. Our results highlight that there are crucial components present in the lung tissue which NTHi require for growth, that are not present in any published variant of SCFM from the Palmer et al. 2007 lineage. Our results may inform future modifications to SCFM recipes to truly mimic the environment of CF lung sputum, and thus, to facilitate study of a wide range of CF pathogens. Data SummaryThe authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files. All raw data has been uploaded to FigShare (https://doi.org/10.6084/m9.figshare.28175300.v1).

microbiology↗

bioRxiv2025

The microbiota of fresh unfermented date palm sap: a culturomics and metagenomics investigation.

Palm sap is an artisanal fermented beverage, the consumption of which has been historically attested for millennia. Despite this, the underlying fermentative microbiota is poorly known. Here, three date palm sap samples collected and bought in Tunisia were investigated for bacteria, methanogenic archaea, and fungi using culturomics and molecular detection techniques, including metagenomics. Metagenomics yielded 250 bacteria, two fungi, and no archaea. Culturomics yielded 36 bacteria, eight fungi, and no archaea. Taken together, these approaches detected 250 bacteria and nine fungi, including 28 bacteria and eight fungi which had been previously unreported in date palm sap, in addition to three potentially new bacteria. Specifically, the bacteria Zymomonas mobilis and Leuconostoc mesenteroides and the fungus Saccharomyces cerevisiae were the most abundant, fermentative species found in the three samples, by culturomics and metagenomics. While determining the source of these microbes of interest was beyond the scope of the study, the data reported here may pave the way for a better understanding of the organic and organoleptic qualities of date palm sap in productive countries. IMPORTANCEThis study provides valuable insights into the microbial composition of date palm sap, a traditional fermented beverage with deep historical roots. Despite its widespread consumption and cultural importance, the microbes driving its fermentation remain largely unexplored. Using advanced methods like metagenomics and culturomics, we uncovered a diverse array of bacteria and fungi, including species crucial for fermentation, such as Zymomonas mobilis, Leuconostoc mesenteroides, and Saccharomyces cerevisiae. Moreover, the discovery of previously unreported microbes highlights the potential for further research into their roles in sap fermentation and quality. Improving our understanding of the microbial communities involved in the fermentation of date palm sap underscores its importance to the field of applied and environmental microbiology.

microbiology↗

bioRxiv2025

Combinatorial effects of bovicin HC5 and epsilon-polylysine against Alicyclobacillus acidoterrestris in orange juice

BACKGROUNDAlicyclobacillus acidoterrestris, a spoilage-causing bacterium in fruit juices remains a concern post-pasteurization, making antimicrobial peptides (AMPs) a promising alternative for its control. Bovicin HC5 and epsilon-polylysine were tested for their antimicrobial efficacy against A. acidoterrestris DSMZ 2498 in both AAM broth and orange juice. This study determined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentration (MBC) for epsilon-polylysine and bovicin HC5 against 106 CFU/mL vegetative cells and activated endospores of A. acidoterrestris DSMZ 2498. Their combined effect was also assessed using the checkerboard method. Viable cell counts were measured after 48 hours at 45 {degrees}C, and thermal resistance of endospores was examined after exposure to both AMPs as well as their combinations, by determining the exposure time (in minutes) required to kill 90 % of the initial population at 95 {degrees}C. Finally, atomic force microscopy (AFM) was employed to investigate their impact on the cell structure and morphology of A. acidoterrestris. RESULTSBovicin HC5 and epsilon-polylysine demonstrated antibacterial effects against A. acidoterrestris DSMZ 2498 inoculated in orange juice. The combination of both AMPs exhibited an additive effect, significantly reducing the number of viable cells after a 48-hour incubation at 45 {degrees}C and a 90 % reduction in thermal resistance at 95 {degrees}C. Atomic force microscopy (AFM) revealed structural and morphological changes in A. acidoterrestris cells treated by these AMPs. CONCLUSIONSThe findings in this study revealed the potential application of bovicin HC5 and epsilon-polylysineas natural preservatives in hurdle technologies to control A. acidoterrestris and improve the microbiological stability and safety of fruit juices.

microbiology↗

bioRxiv2025

A Simple, Low-Cost, and Efficient Protocol for Rapid Isolation of Pathogenic Bacteria from Human Blood

Bacteremia is a serious clinical condition in which pathogenic bacteria enter the bloodstream, putting patients at risk of septic shock and necessitating antibiotic treatment. Choosing the most effective antibiotic is crucial not only for resolving the infection but also for minimizing side effects, such as dysbiosis in the healthy microbiome and reducing the selection pressure for antibiotic resistance. This requires prompt identification of the pathogen and antibiotic susceptibility testing, yet these processes are inherently slow in standard clinical microbiology labs due to reliance on growth-based assays. Although alternative methods exist, they are rarely adopted in clinical settings because they involve complex protocols and high costs for training and infrastructure. Here, we present an optimized, simple protocol for rapidly and efficiently isolating bacterial pathogens from blood without altering typical laboratory workflows. Our method is cost-effective and compatible with commonly available laboratory instruments, offering the advantage of isolating bacterial cells directly, which bypasses the delays associated with traditional blood culture methods and enables faster diagnostic results. The protocol achieved over 70% efficiency within 30 minutes, remained effective at low bacterial concentrations (1-10 bacteria/0.3 mL blood), and preserved bacterial viability with no notable change in growth lag times. We validated the protocol on several clinically relevant bacterial strains, including Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. These findings highlight our protocols potential utility in clinical and research settings, facilitating timely cultures and minimizing diagnostic delays.

microbiology↗

bioRxiv2025

A putative ecocline in Klebsiella pneumoniae

The genetic structure of bacterial species is most often interpreted in terms of demographic processes such as clonal descent but can also reflect natural selection and hence give functional and ecological insight. Klebsiella pneumoniae (KP) disperses effectively around the world and has high recombination rates, which should result in the species having a well-mixed gene pool. Nevertheless, phylogenies based on diverse KP strains contain a "backbone". This structure reflects a component of variation where the first component in Principal Components Analysis (PCA), PC1, explains 16.8% of the total variation. We propose that the component reflects a "bacterial ecocline" generated by diversifying selection on a quantitative genetic trait. We simulated a model in which a trait is influenced by many genes, and strains with the most extreme trait values have a small advantage, which can recapitulate our KP PCA results and other features of its genetic diversity. As well as providing an explanation for the phylogenetic backbone, our results provide insight into how species such as KP can speciate, via stronger selection on the trait or a reduction in gene flow. Our hypothesis that there is a bacterial ecocline in KP raises two questions, namely what the trait is underlying it and why is the trait under diversifying selection? The genes that are most strongly associated with PC1 provide some hints, with the top locus encoding Kpa fimbriae. Identification of the trait, if it exists, should facilitate insight into selection on quantitative genetic traits in natural bacterial populations, which have largely been unstudied in microbiology, except in the atypical context of antibiotic resistance.

microbiology↗

bioRxiv2025

Evaluating Seqstant LiveGene Analysis in Real-Time Assessment of Metagenomic Next-Generation Sequencing (mNGS) Data from Respiratory Samples

BackgroundThe detection of pathogens causing infections by conventional diagnostic methods can be challenging and next-generation sequencing (NGS) technology offers a promising alternative method. In this study, we evaluated the performance of real-time metagenomic next-generation sequencing (rt-mNGS) for the detection of pathogens in respiratory samples. MethodWe used rt-mNGS, using the Seqstant LiveGene Analysis platform, on 335 respiratory samples in comparison to conventional culture results. ResultsWe observed an overall good concordance in 71.64% (240/335) of the methods. The rt-mNGS outperformed the gold standard culture in 16.12% (54/335) of the samples, while the culture was superior in detecting the clinically relevant pathogen in 12.24% (41/335) of the samples. The non-inferiority of rt-mNGS was statistically significant ({delta} = 10, = 0.05, 1 - {beta}= 0.8). We also observed that the real-time analysis of NGS data is beneficial in obtaining reliable timely results as the initial report at cycle 46 exhibits a Positive Predictive Value (PPV) of 93.75% at the species-level with a sensitivity of 32.09%. ConclusionOverall, our study showed the non-inferiority of rt-mNGS compared to the standard-of-care microbiology for respiratory samples with statistical significance. Moreover, the rt-mNGS method exhibited superior sensitivity and superior overall performance. It also uniquely detected certain organisms that are typically hard to culture. However, rt-mNGS reported a higher number of false positives and faced limitations in detecting Aspergillus spp. In conclusion, the study highlights the potential of rt-mNGS as a powerful tool in clinical diagnostics of respiratory infections and beyond.

microbiology↗

bioRxiv2025

An improved catalogue for whole-genome sequencing prediction of bedaquiline resistance in M. tuberculosis using a reproduciblealgorithmic approach.

Bedaquiline (BDQ) has only been approved for use for a little over a decade yet is a key drug for treating multi-drug resistant tuberculosis, however rising levels of resistance threaten to reduce its effectiveness. Catalogues of mutations associated with resistance to bedaquiline are key to detecting resistance genetically for either diagnosis or surveillance. At present building catalogues requires considerable expert knowledge, often requires the use of complex grading rules, and is an irreproducible process. We developed an automated method, catomatic, that associates genetic variants with resistance (or susceptibility) using a two-tailed binomial test with a stated background rate and applied it to a dataset of 11,867 Mycobacterium tuberculosis samples with whole genome and bedaquline susceptibility testing data. Using this framework we investigated how to best classify variants and the phenotypic significance of minor alleles. The genes mmpS5 and mmpL5 are not directly associated with bedaquline resistance, and our catalogue of Rv0678, atpE, and pepQ variants attains a cross-validated sensitivity and specificity of 79.4 {+/-} 1.8 % and 98.5 {+/-} 0.3%, respectively, for 94 {+/-} 0.4% of samples. Identifying samples with subpopulations containing Rv0678 variants improves sensitivity, and detection thresholds in bioinformatic pipelines should therefore be lowered. By using a more permissive and deterministic algorithm trained on a sufficient number of resistant samples we have reproducibly constructed an AMR catalogue for BDQ resistance-associated variants that is comprehensive and accurate. Impact StatementBedaquiline has recently received global endorsement for tuberculosis treatment, yet the genetic determinants of antimicrobial resistance remain incompletely understood. Existing gold-standard methods for building mutation catalogs lack public accessibility and reproducibility. We introduce catomatic, a reproducible and publicly available method that employs simpler statistics to increase sensitivity to resistance-associated variants. This approach has enabled investigations into mechanisms of resistance, the significance of genetic subpopulations, and key data attributes that influence the ease of classifying effects and the accuracy of BDQ resistance phenotype prediction in clinical samples. We strongly emphasise the utility in using reproducible statistics and sustainably developed software in genetics-focussed microbiology.

microbiology↗

bioRxiv2025

Microbial ecology of acidic, biogenic gypsum: Community structure and distribution of extremophiles on freshly formed and relict sulfate deposits in a hydrogen sulfide-rich cave

Sulfate minerals are abundant on the Martian surface, and many of these evaporite deposits are thought to have precipitated from acidic fluids. On Earth, gypsum (CaSO4*2H2O) and other sulfates sometimes form under acidic conditions, so exploring the extremophilic life that occurs in these mineral environments can help us evaluate the astrobiological potential of acid sulfate depositional settings. Here, we characterized the microbial communities associated with acidic gypsum deposits in a sulfuric acid cave, where sulfate precipitation is driven by sulfide-oxidizing bacteria and archaea. We used 16S rRNA gene sequencing and cell counts to characterize gypsum-associated microorganisms in freshly formed and relict deposits throughout the cave, in order to test hypotheses about how microbial community composition and abundance would vary with distance from the sulfidic water table and with the concentration of H2S(g) and other gases in the cave atmosphere. We found that actively-forming gypsum in the lower cave levels was colonized by low diversity communities of sulfide-oxidizing chemolithotrophs and other acidophiles that have few cells compared to other environments in the cave. The most abundant taxa were Acidithiobacillus, Metallibacterium, Mycobacteria, and three different Thermoplasmatales-group archaea, which occupied distinct niches based on proximity to sulfidic streams and the concentration of gases in the cave air. In contrast, deposits in older cave levels had more diverse communities that are dominated by chemoorganotrophic and methanotrophic taxa. These findings show that acidic sulfate deposits serve as habitats for extremophilic microorganisms, and broaden our knowledge of the life associated with terrestrial sulfates. ImportanceGypsum and other sulfate salts are common on Mars, and many of these deposits are thought to have formed from acidic fluids early in the planets history. Understanding the life that survives and thrives in similar environments on Earth is therefore crucial for evaluating whether these Martian sulfates are or ever were habitable. One such environment where acidic gypsum occurs is in sulfuric acid caves, where extremophilic microorganisms drive the precipitation of sulfate minerals by oxidizing hydrogen sulfide gas from the cave atmosphere. Here, we characterized the communities of microorganisms on freshly formed and ancient gypsum in the Frasassi Caves, and found that the gypsum deposits hosted microbial communities that changed based on chemical energy availability and the age of the gypsum. Our findings underscore the importance of chemical and microbiological interactions in shaping habitable niches, and provide context for searching for past or present life in acidic Martian sulfates.

microbiology↗

bioRxiv2025

Calcite Precipitation by a Nitrogen-Fixing Cyanobacterium

Microbiologically induced calcium carbonate precipitation (MICP) is the process through which the metabolic activity of microorganisms causes the precipitation of calcium carbonate, which can result in solidification of sediment. In cyanobacteria, MICP is thought to occur primarily because cells sequester bicarbonate for the photosynthetic process, thereby lowering the pH of the surrounding media. However, these mechanisms are still poorly understood. Here we show direct evidence of MICP caused by the filamentous cyanobacterium Anabaena. Anabaena differentiates into photosynthetic vegetative cells and nitrogen-fixing heterocysts. Using quantitative microscopy, we show that MICP occurs due to two distinct mechanisms: Firstly, mechanical stress on vegetative cells can cause leakage and/or lysis, releasing sequestered bicarbonate into the environment, resulting in formation of new crystals. Secondly, contact between a heterocyst and a calcite crystal seed appears to cause rapid crystal growth. Our results suggest an evolutionary benefit of contact-mediated precipitation to anchor cyanobacteria growing in tidal regions. By providing greater insight into MICP caused by Anabaena, these results could be used to optimize bio-cement production, thereby enabling a green construction material that could assist with carbon sequestration and reducing the impact of climate change.

microbiology↗