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bioRxiv2021

Ancient and modern geochemical signatures in the 13,500-year sedimentary record of Lake Cadagno

Although lake sediments are globally important organic carbon sinks and therefore important habitats for deep microbial life, the deep lacustrine biosphere has thus far been little studied compared to its marine counterpart. To investigate the impact of the underexplored deep lacustrine biosphere on the sediment geochemical environment and vice versa, we performed a comprehensive microbiological and geochemical characterization of a sedimentary sequence from Lake Cadagno covering its entire environmental history since formation following glacial retreat. We found that both geochemical gradients and microbial community shifts across the [~]13.5 kyr subsurface sedimentary record reflect redox changes in the lake, going from oxic to anoxic and sulfidic. Most microbial activity occurs within the top 40 cm of sediment, where millimolar sulfate concentrations diffusing in from the bottom water are completely consumed. In deeper sediment layers, organic carbon remineralization is much slower but microorganisms nonetheless subsist on fermentation, sulfur cycling, metal reduction, and methanogenesis. The most surprising finding was the presence of a deep, oxidizing groundwater source. This water source generates an inverse redox gradient at the bottom of the sedimentary sequence and could contribute to the remineralization of organic matter sequestered in the energy-limited deep subsurface.

microbiology↗

bioRxiv2021

Development and Validation of a MALDI-TOF-Based Model to Predict Extended-Spectrum Beta-Lactamase and/or Carbapenemase-Producing in Klebsiella pneumoniae Clinical Isolates

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS) is a reference method for microbial identification and it can be used to predict Antibiotic Resistance (AR) when combined with artificial intelligence methods. However, current solutions need time-costly preprocessing steps, are difficult to reproduce due to hyperparameter tuning, are hardly interpretable, and do not pay attention to epidemiological differences inherent to data coming from different centres, which can be critical. We propose using a multi-view heterogeneous Bayesian model (KSSHIBA) for the prediction of AR using MALDI-TOF MS data together with their epidemiological differences. KSSHIBA is the first model that removes the ad-hoc preprocessing steps that work with raw MALDI-TOF data. In addition, due to its Bayesian probabilistic nature, it does not require hyperparameter tuning, provides interpretable results, and allows exploiting local epidemiological differences between data sources. To test the proposal, we used data from 402 Klebsiella pneumoniae isolates coming from two different domains and 20 different hospitals located in Spain and Portugal. KSSHIBA outperforms current state-of-the-art approaches in antibiotic susceptibility prediction, obtaining a 0.78 AUC score in Wild Type classification and a 0.90 AUC score in Extended-Spectrum Beta-Lactamases (ESBL)+Carbapenemases (CP)-producers. The proposal consistently removes the need for ad-hoc preprocessing by working with raw MALDI-TOF data, which, in turn, reduces the time needed to obtain the results of the resistance mechanism in microbiological laboratories. The proposed model implementation as well as both data domains are publicly available.

microbiology↗

bioRxiv2021

Bacteriocinogenic lactic acid bacteria in the traditional cereal-based beverage Boza: a genomic and functional approach

Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as P. acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 AU/ml to 12800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37{degrees}C. All bacteriocins remained active after incubation at pH 2.0 to 10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage {beta}-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining metagenomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.

microbiology↗

bioRxiv2021

Geological processes mediate a subsurface microbial loop in the deep biosphere

Summary paragraphThe deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores1,2. Whereas dormancy and survival at theoretical energy minima are hallmarks of subsurface microbial populations3, the roles of fundamental ecological processes like dispersal and selection in these environments are poorly understood4. Here we combine geophysics, geochemistry, microbiology and genomics to investigate biogeography in the subsurface, focusing on bacterial endospores in a deep-sea setting characterized by thermogenic hydrocarbon seepage. Thermophilic endospores in permanently cold seabed sediments above petroleum seep conduits were correlated with the presence of hydrocarbons, revealing geofluid-facilitated cell migration pathways originating in deep oil reservoirs. Genomes of thermophilic bacteria highlight adaptations to life in anoxic petroleum systems and reveal that these dormant populations are closely related to oil reservoir microbiomes from around the world. After transport out of the subsurface and into the deep-sea, thermophilic endospores re-enter the geosphere by sedimentation. Viable thermophilic endospores spanning the top several metres of the seabed correspond with total endospore counts that are similar to or exceed the global average. Burial of dormant cells enables their environmental selection in sedimentary formations where new petroleum systems establish, completing a geological microbial loop that circulates living biomass in and out of the deep biosphere.

microbiology↗

bioRxiv2021

Factors Influencing the Detection of Antibacterial Resistant Escherichia coli in Faecal Samples from Individual Cattle

AimsTo investigate whether on-farm antibacterial usage (ABU), environmental antibacterial resistant (ABR) Escherichia coli prevalence, and sampling and sample handling methodologies are associated with ABR E. coli positivity in individual faecal samples from dairy heifers. Methods and ResultsThree hundred and sixty-four heifers from 37 farms were sampled via rectal or faecal pat sampling. Samples were stored at -80{degrees}C for variable periods before microbiological analysis. Data analysis was through a multilevel, multivariable logistic regression approach. Individual rectal samples had increased odds of positivity for amoxicillin, cefalexin and tetracycline-resistant E. coli. Sample storage for 6-12 month was associated with decreased odds of finding amoxicillin and tetracycline-resistant E. coli. On-farm ABU had little influence, and environmental ABR E. coli prevalence had no significant influence on on the odds of sample-level positivity for ABR E. coli. ConclusionsSampling methodology and sample handling have a greater association than on-farm factors with the detection of ABR E. coli in individual faecal samples from dairy heifers. Significance and Impact of the StudySampling and storage methodologies should be considered carefully at the point of designing ABR surveillance studies in livestock and their environments and, where possible, standardised between and within future studies.

microbiology↗

bioRxiv2021

A novel growth and isolation medium for exoelectrogenic bacteria

A microbiological isolation and growth medium that can effectively discriminate electrochemically active exoelectrogenic bacteria from other non-exoelectrogenic bacteria, is currently unavailable. In this study, we developed a novel chromogenic growth and isolation solid medium based on MnO2 that can selectively allow the growth of exoelectrogenic bacteria and change the medium colour in the process. Known exoelectrogenic bacteria such as Shewanella oneidensis MR1 and other such bacteria from functional microbial fuel cell (MFC) anodes were capable of growing and changing colour in the novel growth medium. On the contrary, non-exoelectrogenic bacteria such as Escherichia coli ATCC 25922 were incapable of growing and inducing a colour change in the novel medium. Further biochemical characterisation of these isolated exoelectrogenic bacteria by Raman micro-spectroscopy demonstrated that these bacteria over express cytochrome proteins that are vital in extracellular electron transfer events. This medium is a convenient method to isolate exoelectrogenic bacteria from complex environmental samples.

microbiology↗

bioRxiv2021

FIRST REPORT OF HALOBACTERIA DOMINANCE IN A TROPICAL CAVE MICROBIOME

Scarce studies on microbial diversity in tropical caves have been published, a subterranean system still neglected from a microbiological point of view. Although most published studies are about temperate caves, usually archaeas and fungi have less attention than bacterial communities. Here, the microbiome structure and composition in a tropical cave system, as well the main environmental drivers, were studied during the wet and dry season. Soil and sediments from three different habitats at the cave (surface, entrance cave and dark zone) were sampled. Samples were characterized (temperature, air and substrate humidity, salinity, pH, nitrogen and organic carbon content, and chemical composition) and the microbiome was assessed by high-throughput sequencing, using amplicon sequencing (16S and ITS). Prokaryotic communities were dominated by Halobacteria, Actinobacteria and Bacilli, while fungal communities showed high abundance of Sordariomycetes. Microbiomes from the cave entrance, where a significantly elevated salinity levels were found, supported up to 63% of Haloarchaea compared to the other habitats studied. Differences in community structure were significant between habitats, but no influence of the season was observed. Main environmental drivers of community assembly included nitrogen and organic carbon content, temperature, and salinity. This is the first report of Halobacteria dominance in cave habitats, where they likely play important roles in nitrogen and phosphorus cycles. The cave entrance had lower diversity, but higher degree of microbial endemism, which characterize it as an important cave ecotone. The prevalence of heterotrophic microbial groups implies trophic structure based on detritivores, particularly in the dark zones. Our study brings new insights on microbiome composition in the underexplored tropical cave habitats.

microbiology↗

bioRxiv2021

Mechanical ventilation affects the microecology of the rat respiratory tract

BackgroundThe most common second strike in mechanically ventilated patients is a pulmonary infection caused by the ease with which bacteria can invade and colonize the lungs due to mechanical ventilation. At the same time, metastasis of lower airway microbiota may have significant implications in the development of intubation mechanical ventilation lung inflammation. Thus, we establish a rat model of tracheal intubation with mechanical ventilation and explore the effects of mechanical ventilation on lung injury and microbiological changes in rats. MethodsSprague-Dawley rats were randomized into control, Spontaneously Breathing (1, 3, 6 hours), Mechanical ventilation(1, 3, 6 hours) groups. Lung wet to dry weight ratio (W/D weight ratio) and Lung histopathological injury score were evaluated.16SrDNA sequencing was performed to explore respiratory flora changes. ResultsBacterial diversity was comparable between healthy and intubation mechanical ventilation rats, with time relation. Ordination analyses revealed that samples clustered more dispersing by tracheal intubation and mechanical ventilation. Finally, predicted metagenomes suggested a substantial increase in biofilm formation phenotype during early tracheal intubation and mechanical ventilation. ConclusionCollectively, these results establish a link between the duration of mechanical ventilation and alterations to the respiratory tract microecology. In future studies, we hope to discover the effectiveness of new immunomodulatory or probiotic bacteria to prevent airway diseases associated with ventilator therapy.

microbiology↗

bioRxiv2022

Survival and Transcriptomic Response of Clinically relevant pathogens on Surfaces in Relation to Probiotic and Traditional Cleaning Strategies

Indoor surfaces are paradoxically presumed to be both colonized by pathogens, necessitating disinfection, and "microbial wastelands." In these resource-poor, dry environments, competition and decay are thought to be important drivers of microbial community composition. However, the relative contributions of these two processes have not been specifically evaluated. To bridge this knowledge gap, we used microcosms to specifically evaluate whether interspecies interactions occur on surfaces. We combined transcriptomics and traditional microbiology techniques to investigate whether competition occurred between two clinically important pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, and a probiotic cleaner containing a consortium of Bacillus species. Probiotic cleaning seeks to take advantage of ecological principles such as competitive exclusion, thus using benign microorganisms to inhibit viable pathogens, but there is limited evidence that competitive exclusion in fact occurs in environments of interest (i.e., indoor surfaces). Our results indicate that competition in this setting has a negligible impact on community composition but may influence the functions expressed by active organisms. Although Bacillus spp. remained viable on surfaces for an extended period of time after application, viable colony forming units (CFUs) of A. baumannii recovered following exposure to a chemical-based detergent with and without Bacillus spp. showed no statistical difference. Similarly, for K. pneumoniae, there were small statistical differences in CFUs between cleaning scenarios with or without Bacillus spp. in the chemical-based detergent. The transcriptome of A. baumannii with and without Bacillus spp. exposure shared a high degree of similarity in overall gene expression, but the transcriptome of K. pneumoniae differed in overall gene expression, including reduced response in genes related to antimicrobial resistance. Together, these results highlight the need to fully understand the underlying biological and ecological mechanisms for community assembly and function on indoor surfaces, as well as having practical implications for cleaning and disinfection strategies for infection prevention.

microbiology↗

bioRxiv2022

Benchmarking taxonomic classifiers with Illumina and Nanopore sequence data for clinical metagenomic diagnostic applications

Culture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of ten common bloodstream pathogens and six frequent blood culture contaminants (n=1600). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken, and Centrifuge, utilising mini (8GB) and standard (30-50GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 98.46% (IQR 93.0:99.3) [range 57.1:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 79.3% (IQR 39.1:88.8) [range 11.2:100]. Classification performance varied by species, with E. coli being more challenging to classify correctly (59.4% to 96.4% reads with correct species, varying by tool used). By filtering out shorter Nanopore reads (<3500bp) we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. This "bioinformatic contamination" should be accounted for in metagenomic pipelines to ensure accurate results that can support clinical decision making. ImportanceMetagenomics may transform clinical microbiology by enabling more rapid species detection in a potentially unbiased manner and reducing reliance on culture-based approaches. However, it is still limited by ongoing challenges such as sequencing and classification software errors. In this study, we use simulated and real-world data to define the intrinsic rates of species misclassification that occur using Illumina and Oxford Nanopore sequencing platforms with commonly used taxonomic classification tools and databases. We quantify the extent of "bioinformatic contamination" arising from the classification process. This enables us to identify the best performing tools that maximize classification accuracy, and to suggest how taxonomic misclassification can be formally accounted for in clinical diagnostic workflows. Specifically, we specify thresholds for identifying or excluding polymicrobial infections in metagenomic samples, based on rates of misclassification of similar species, which might have clinical implications when treating infection.

microbiology↗

bioRxiv2022

Meta-analysis of the black soldier fly (Hermetia illucens) microbiota based on 16S rRNA gene amplicon sequencing

Black soldier fly larvae (BSFL) belong to the most widely reared insect species as an alternative protein source at industrial scale. Bacteria in the larval gut can provide benefits for the animal, though some bacteria can also be pathogenic for the insect. Accurate characterization of the BSFL microbiota is important for the production of BSFL in terms of yield and microbiological safety. In this study, 16S ribosomal RNA gene sequence data sets from 11 studies were re-analysed to gain better insights in the BSFL gut microbiota, potential factors that influence their composition, and differences between the gut and the whole larvae microbiota. A core gut microbiota was found consisting of members of Enterococcus, Klebsiella, Morganella, Providencia, and Scrofimicrobium. Further, the factors "Study", "Age" and "Feed" significantly affected the microbiota gut composition. When compared to whole larvae, a significantly lower number of observed zero-radius Operational Taxonomic Units and a lower diversity was found for gut samples, suggesting that the larvae harboured additional microbes on their cuticle or in the insect body. Universal choices in insect sample type, primer selection and bio-informatics can strengthen future meta-analyses and improve our understanding of the BSFL gut microbiota towards the optimization of insect production.

microbiology↗

bioRxiv2022

Exit from lag phase in populations of Pseudomonas fluorescens is determined by strong interactions among cells

The relationship between the number of cells colonizing a new environment and time for resumption of growth is a subject of long-standing interest. In microbiology this is known as the "inoculum effect". Its mechanistic basis is unclear with possible explanations ranging from the independent actions of individual cells, to collective actions of populations of cells. Progress requires precise measurement of lag time distributions while at the same time, experimentally controlling inoculum size. Here we use a millifluidic droplet device in which the growth dynamics of hundreds of populations founded by different numbers of Pseudomonas fluorescens cells, ranging from a single cell, to one thousand cells, were followed in real time. Our data show that lag phase decreases with inoculum size. The average decrease, variance across droplets, and distribution shapes, follow predictions of extreme value theory, where the inoculum lag time is determined by the minimum value sampled from the single-cell distribution. Our experimental results show that exit from lag phase depends on strong interactions among cells, consistent with a "leader-cell" triggering end of lag phase for the entire population.

microbiology↗

bioRxiv2022

Micro-colony observation: A rapid and simple approach to count bacterial colony forming units

For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFU) has always been the preferred method in microbiology owing to its simplicity, albeit laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing micro-colonies in conjunction with the perks of a microscope. Here, we employed a simple method of five microliter spotting of differently diluted bacterial culture multiple times on a single Petri dish followed by finding out CFU by counting micro-colonies using a phase-contrast microscope. In this method, the CFU of an Escherichia coli culture can be estimated within a four-hour period. Further, within a ten-hour period, CFU in a culture of Ralstonia solanacearum, a bacterium with a generation time of around 2 h, can be estimated. The CFU number determined by micro-colonies observed is comparable with that obtained by the dilution plating method. Micro-colonies number observed in the early hours of growth (2 h in case of E. coli and 8 h in case of R. solanacearum) were found to remain consistent at later hours, though there was a noticeable increase in the size of the colonies. It suggested that micro-colonies observed in the early hours indeed represent the bacterial number in the culture. Practical applications to this counting method were employed in studying the rifampicin-resistant mutation rate as well as performing the fluctuation test in E. coli. The method described here results in a 90% reduction of labour, time and resources. Thus, the method is likely to be adopted by many microbiologists in their routine laboratory research.

microbiology↗

bioRxiv2022

The efficacy of commercial decontamination agents differs between standardised test settings and real-world usage for a variety of bacterial species

Decontamination of surfaces and items plays an important role in reducing the spread of infectious microorganisms in many settings including hospitals and research institutes. Regardless of the location, appropriate decontamination procedures are required for maintaining biosafety and biosecurity. For example, effective decontamination of microbial cultures is essential to ensure proper biocontainment and safety within microbiological laboratories. To this end, many commercial decontamination agents are available which have been tested to a prescribed standard to substantiate their efficacy. However, these standardised tests are unlikely to accurately reflect all conditions encountered in medical and biomedical research. Despite this, laboratory workers and other users of decontamination agents may assume that all decontamination agents will work in all situations. We tested commonly used commercial decontamination agents against a range of bacterial species to determine their efficacy under real-world laboratory conditions. As each decontamination agent has a different recommended dilution for use, to compare their efficacy we calculated their effective ratio which reflects the difference between the manufacturer-recommended dilution and the dilution needed to achieve decontamination under real-world laboratory conditions. Effective ratios above 1 indicate that the agent was active at a dilution more dilute than recommended whereas effective ratios lower than 1 indicate that the agent required a higher concentration than recommended. Our results show that the quaternary ammonium agents TriGene Advance and Chemgene HLD4L were the most active out of the agents tested, with biocidal activity measured at up to 64 times the recommended dilution. In contrast, hypochlorite (bleach) and Prevail (stabilised hydrogen peroxide) had the lowest effective ratios amongst the tested agents. In conclusion, our data suggest that not all decontamination agents will work at the recommended dilutions under real-world laboratory conditions. We recommend that the protocols for the use of decontamination agents are verified under the specific conditions required to ensure they are fit for purpose.

microbiology↗

bioRxiv2022

Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments

Coastal zones account for significant global marine methane emissions to the atmosphere. In coastal ecosystems, the tight balance between microbial methane production and oxidation in sediments prevents most methane from escaping to the water column. Anthropogenic activities, causing eutrophication and bottom water deoxygenation, could disrupt this balance in the microbial methane cycle and lead to increased methane release from coastal sediments. Here, we combined microbiological and biogeochemical analyses of sediments from three sites along a bottom water redox gradient (oxic-hypoxic-euxinic) in the eutrophic Stockholm Archipelago to investigate the impact of anthropogenically-induced redox shifts on microbial methane cycling. At both the hypoxic and euxinic site, sediments displayed a stronger depletion of terminal electron acceptors at depth and a shoaling of the sulfate-methane transition zone in comparison to the oxic site. Porewater methane and sulfide concentrations and potential methane production rates were also higher at the hypoxic and euxinic site. Analyses of metagenome-assembled genomes and 16S rRNA gene profiling indicated that methanogens became more abundant at the hypoxic and euxinic site, while anaerobic methane-oxidizing archaea (ANME), present in low coverage at the oxic site, increased at the hypoxic site but virtually disappeared at the euxinic site. A 98% complete genome of an ANME-2b Ca. Methanomarinus archaeon had genes encoding a complete reverse methanogenesis pathway, several multiheme cytochromes, and a sulfite reductase predicted to detoxify sulfite. Based on these results, we infer that sulfide exposure at the euxinic site led to toxicity in ANME, which, despite the abundance of substrates at this site, could no longer thrive. These mechanistic insights imply that the development of euxinia, driven by eutrophication, could disrupt the coastal methane biofilter, leading to increased benthic methane release and potential increased methane emissions from coastal zones to the atmosphere.

microbiology↗

bioRxiv2022

Is it the same strain? Defining genomic epidemiology thresholds tailored to individual outbreaks

BackgroundEpidemiological surveillance relies on microbial strain typing, which defines genomic relatedness among isolates to identify case clusters and their potential sources. No consensus exists on the choice of thresholds of genomic relatedness to define clusters. While a priori defined thresholds are often applied, outbreak-specific features such as pathogen mutation rate and duration of source contamination should be considered. MethodsWe developed a forward model of bacterial evolution to simulate mutation within a population diversifying at a specific mutation rate, with specific outbreak duration and sample isolation dates. Based on the resulting expected distribution of genetic distances we define a threshold beyond which isolates are considered as not part of the outbreak. We additionally embedded the model into a Markov Chain Monte Carlo inference framework to estimate, from data including sampling dates or isolates genetic variation, the most credible mutation rate or time since source contamination. FindingsA simulation study validated the model over realistic durations and mutation rates. When applied to 16 published datasets describing foodborne outbreaks, our framework consistently identified outliers. Appropriate thresholds for grouping cases were obtained for 14 outbreaks. For the remaining two outbreaks, re-estimation of the duration of outbreak lead to updated threshold values and was more likely, given our model, to result in the observed genetic distances. InterpretationWe propose an evolutionary approach to the single strain conundrum by defining the genetic threshold based on individual outbreak properties. The framework provides an informed estimation of the likelihood of a cluster given the samples epidemiological and microbiological context. This forward model, applicable to foodborne or environmental-source single point case clusters or outbreaks, will be useful for epidemiological surveillance and to guide control measures. FundingThis work was supported financially by the MedVetKlebs project, a component of European Joint Programme One Health EJP, which has received funding from the European Unions Horizon 2020 research and innovation programme under Grant Agreement No 773830. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for studies published between database inception and April 3, 2021, with the term (threshold OR cut-off OR genetic relatedness) AND (outbreak) AND (cgMLST OR wgMLST OR SNPs) AND (microbial OR bacteria OR bacterial OR pathogen). We found 222 related articles. Most studies define a fixed SNP threshold that relate outbreak strains based on previous observations. One original study identifies outbreak clusters based on transmission events. However, it relies on strong assumptions about molecular clock and transmission processes. Added value of this studyOur study describes a new method based on a forward Wright-Fisher model to find the most credible genetic distance threshold. This method is fast and simple to use with only few assumptions, informed by outbreak duration and pathogen mutation rate. By using SNP or cgMLST pairwise distances and sample collection dates of the outbreak of interest, the algorithm provides context-based guidance to separate outbreak strains from outliers. Implications of all the available evidenceThe fast and easy method developed here enables to move away from a priori defined thresholds. Defining clusters more accurately based on the specific features of outbreaks, and the ability to estimate outbreak duration, will provide the needed precision for epidemiological surveillance and should contribute to leverage molecular epidemiology data more efficiently for the purpose of uncovering contamination sources. Data Availability StatementAll data and code used for this manuscript is available online at https://gitlab.pasteur.fr/BEBP.

microbiology↗

bioRxiv2022

Mucosal microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients

Perturbations in the gut microbiome have been linked to the promotion and prognosis of colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite the increasing health burden inflicted by CRC in low- and middle-income countries like Vietnam, the CRC-specific microbiome in these populations remains underexplored. Here we conducted a study in Vietnam to enrol 43 CRC patients (cases) and 25 patients with non-cancerous colorectal polyps (controls) between December 2018 and January 2020. Our study investigated the mucosal microbiome signature and genomic diversity of Fusobacterium in Vietnamese CRC patients, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome sequencing. We found that several oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour mucosa, and these two bacteria were also more enriched in tumours of advanced CRC stages (III-IV). We obtained 53 Fusobacterium genomes from the saliva, tumour and non-tumour mucosa of six CRC patients. Isolates from the gut mucosa belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of F. periodonticum. The Fusobacterium population within each individual was distinct and in many cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within each individual, tumour-associated Fusobacterium were clonal to those isolated from non-tumour mucosa, but distantly related to those isolated from saliva. Genes encoding major virulence factors (Fap2 and RadD) showed variability in length and evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.

microbiology↗

bioRxiv2022

Cell Chromatography: Biocompatible chromatographic separation and interrogation of microbial cells

The isolation of pure, single colonies lies at the heart of experimental microbiology. However, a microbial colony typically contains around one million cells at all stages of the life cycle. Here we describe a novel, cell chromatography method that facilitates the capture, purification and interrogation of microbial cells from both single and mixed cultures. The method described relies on, but is not limited to, differences in surface charge to separate bacterial strains. The method is fully biocompatible, leading to no significant loss of cell viability. The chromatographic capture of cells, combined with selective elution methods facilitates a greater level of experimental control over the sample inputs required for downstream high throughput and high sensitivity, analytical methods. The application of the method for interrogating the antibiotic resistance of bacterial strains and for the separation of bacteria from environmental samples is illustrated.

microbiology↗