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bioRxiv2023

Analyzing bacterial community compositions by GroEL-taxoproteomics

Profiling bacterial populations in mixed communities is a common task in microbiology. 16S rRNA-based sequencing is a widely accepted and functional approach but relies on amplification primers and cannot quantify isotope incorporation. Tandem mass spectrometry proteotyping is an effective alternative for taxonomically profiling microorganisms. We suggest that targeted proteotyping approaches can complement traditional population analyses. Therefore, we describe an approach to assess bacterial community compositions at the family level using the taxonomic marker protein GroEL, which is ubiquitously found in bacteria, except few obligate intracellular species. We refer to our method as GroEL-proteotyping. GroEL-proteotyping is based on high-resolution tandem mass spectrometry of GroEL peptides and identification of GroEL-derived taxa via a Galaxy workflow and a subsequent Python-based analysis script. Its advantage is that it can be performed with a curated and extendable sample-independent database and that GroEL can be pre-separated by SDS-PAGE to reduce sample complexity, improving GroEL identification while simultaneously decreasing the instrument time. GroEL-proteotyping was validated by employing it on a comprehensive raw data set obtained through a metaproteome approach from synthetic microbial communities as well as real human gut samples. Our data show that GroEL-proteotyping enables fast and straightforward profiling of highly abundant taxa in bacterial communities at reasonable taxonomic resolution.

microbiology↗

bioRxiv2023

Bacterial risk evaluation of widely consumed fast foods by the younger population in Bangladesh and its potential health effects

Food-borne illness is a significant public health concern in Bangladesh, especially among the younger generation. They often consume spicy street foods, such as Fusca, Hog plum bedfellow, and Chotpoti. These foods can be contaminated with harmful bacteria, which can cause various diseases, including diarrhea, cholera, and dysentery. This study investigated the microbiological quality of three commonly consumed street foods in Bangladesh. We collected samples of Fusca, Hog plum bedfellow, and Chotpoti from different vendors in Dhaka and analyzed them in a laboratory. We found that all three of the food samples were contaminated with a variety of bacteria, including Bacillus spp., Haemophilus spp., Salmonella spp., Klebsiella spp., Staphylococcus spp., and Streptococcus spp. Some of these bacteria, such as Haemophilus spp., were resistant to the antibiotic ciprofloxacin. Our findings suggest that there is a high risk of food-borne illness associated with the consumption of street foods in Bangladesh. We urge vendors and consumers to take steps to improve food safety, such as proper food handling and cooking and clean water and utensils.

microbiology↗

bioRxiv2023

HIV-Tocky system to visualize proviral expression dynamics

The stably integrated pool of HIV-1 proviruses in the host genome stands against curative strategies. This reservoir is extremely heterogeneous with respect to host cell type, anatomical location, integration site, and replication fitness. During the initial phase of infection, only a few infected cells can resist host immune clearance or cytopathic effect and establish this resistant pool. The mechanisms underlying HIV latency initiation are not fully resolved yet. In the current study, we propose and validate a new reporter model for monitoring HIV-1 provirus silencing and reactivation using Timer of cell kinetics and activity (Tocky). HIV-Tocky system uses a fluorescent Timer protein whose emission spectrum spontaneously shifts from blue to red to reveal HIV-1 provirus dynamics. We dissected provirus transcriptional phases into early, persistent, recently silenced, and latent. To our knowledge, this is the first report to distinguish two latent subsets: a directly non-expressing population and a recently silenced after brief expression. In-depth integration site analysis suggested that the distribution of proviruses in directly latent cells was similar to that in actively transcribing cell population, whereas recently silenced cells tended to harbor proviruses integrated into heterochromatin. Furthermore, we established a library of various single integration clones at which we utilized to demonstrate the efficiency of the block-and-lock strategy by capturing the fast dynamics of silencing that were overlooked in previous models. In summary, we propose HIV-Tocky system to serve as a time-sensitive model that can capture the dynamics of provirus expression, making it a useful tool for HIV latency research. Significance StatementDeterminants of HIV-1 latency establishment are yet to be elucidated. This reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allowed; for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies. ClassificationBiological Sciences, Microbiology.

microbiology↗

bioRxiv2023

Efflux pump mutations in Pseudomonas aeruginosa cause low-level clinical resistance and high-level tolerance to antibiotics in patients

Pseudomonas aeruginosa is one of the microorganisms with high-risk regarding antimicrobial resistance, since it has an overwhelming capacity to acquire antibiotic resistance, mainly by mutations during persistent lung infections. Mutations in mexZ, encoding the local negative regulator of genes encoding the MexXY efflux pump, are very frequently acquired at early stages of P. aeruginosa infections, while they are rarely selected for in vitro. Although traditionally related to resistance to the first-line drug tobramycin, caused by the overproduction of the aminoglycosides MexXY efflux pump, mutations in mexZ are actually associated with low levels of aminoglycosides resistance when determined in the clinical microbiology laboratory. This very moderate but frequent phenotype suggests that these mutations may shape the infection process, beyond causing conventional resistance. Here we investigated the colonization strategy of a mexZ mutant, compared to a wild-type strain, in a human airway infection model. We observed that the mexZ mutant tends to accumulate inside the epithelial cell layer. This behaviour allows bacteria to colonise the epithelium while being more protected against diverse antibiotics. The altered colonization phenotype was caused by the overexpression of lecA, a Quorum Sensing regulated gene encoding a lectin involved in P. aeruginosa tissue invasiveness. lecA upregulation was underlied by the competition for the shared porin, OprM, between the overproduced MexXY and the MexAB efflux pump, responsible for extruding Quorum Sensing molecules. These findings suggest that standardised antimicrobial susceptibility determined in the clinic may be misleading because antibiotic resistance often depends on the infection environment.

microbiology↗

bioRxiv2023

Unveiling Local Radiation Events through Metagenome Assembled Genomes of Archaea and Bacteria in Hypersaline Microbial Mats from the Archean Domes Site, Cuatro Cienegas, Coahuila, Mexico

A comprehensive study was conducted in the Cuatro Cienegas Basin (CCB) in Coahuila, Mexico, known for its remarkable microbiological diversity and unique physicochemical properties. The ''Archaean Domes '' (DA) in the CCB are hypersaline, non-lithifying microbial mats. This study focused on analyzing the small domes and circular structures formed in DA through metagenome assembly genomes (MAGs) with the aim of finding new microorganisms and providing information on the tree of life in a place as diverse as the CCB. In total, 329 MAGs were identified, including 52 archaea and 277 bacteria. Remarkably, 30 Archaea and 154 Bacteria could not be classified at the genus level, highlighting the remarkable diversity of CCB. The CCBs showed significant diversity at the phylum level, with Proteobacteria being the most abundant, followed by Euryarchaeota, Firmicutes, Bacteroidetes, Actinobacteria, Cyanobacteria, Spirochaetes, Chloroflexi, Planctomycetes, Candidatus Parvarchaeota, Verrucomicrobia, Balneolaeota, Nitrospirae, and Tenericutes. Subsequently, the MAGs were classified into a phylogenetic tree. In Archaea, monophyletic groups MAGs belonged to the phyla Archaeoglobi, Candidatus Aenigmarchaeota, Candidatus Nanoarchaeota, Candidatus Lokiarchaeota, and Halobacteriota. Among the Bacteria, monophyletic groups were identified as well, including Spirochaetes, Proteobacteria, Planctomycetota, Actinobacteria, Verrucomicrobiota, Bacteroidetes, Bipolaricaulota, Desulfobacterota, and Cyanobacteria. These monophyletic clusters may indicate radiation events that are likely influenced by geographical isolation as well as extreme environmental conditions reported in AD pond like phosphorus deficiency (122:42:1 C:N:P), fluctuating pH and a salinity of 5.28%

microbiology↗

bioRxiv2023

Preventing E. coli biofilm formation with antimicrobial peptide surface coatings: recognizing the dependence on the bacterial binding mode using live-cell microscopy

Antimicrobial peptides (AMPs) can kill bacteria by destabilizing their membranes, yet, to translate these molecules properties into a covalently attached coating is challenging. Standard microbiology methods do not work well for grafted AMPs, particularly it is difficult to distinguish the AMPs bactericidal potency from factors relating to bacterias binding behavior, e.g., which type of and how persistent bacteria-surface contacts that is necessary. Here we present a method combining live-cell microscopy and microfluidics to study the response of E. coli challenged by the same small AMP either in solution or grafted to the surface through click chemistry. The AMP coating initially suppressed bacterial growth as strongly as AMPs in solution. While AMPs in solution eventually killed the E. coli bacteria, those binding to the AMP coating changed contact mode one hour after binding and then became insensitive to it. The transition depended on binding-induced expression of Type 1 fimbriae, which limits contact between the AMPs and the E. coli outer membrane. By quantifying several different factors contributing to the antibacterial efficacy, these measurements provide a holistic understanding of how antibacterial surface coatings function. We therefore expect this tool to be important for the design of elaborate antibacterial coatings that can reduce the need for antibiotics and thus contribute to slower spreading of antibiotic resistance genes.

microbiology↗

bioRxiv2023

Long-term history dependence of growth rates of E. coli after nutrient shifts

According to a widely accepted paradigm of microbiology, steady-state growth rates are determined solely by current growth conditions1-3 and adaptations between growth states are rapid, as recently recapitulated by simple resource allocation models4. However, even in microbes overlapping regulatory networks can yield multi-stability or long-term cellular memory. Species like Listeria monocytogenes5 and Bacillus subtilis "distinguish" distinct histories for the commitment to sporulation6, but it is unclear if these states can persist over many generations. Remarkably, studying carbon co-utilization of Escherichia coli, we found that growth rates on combinations of carbon sources can depend critically on the previous growth condition. Growing in identical conditions, we observed differences in growth rates of up to 25% and we did not observe convergence of growth rates over 15 generations. We observed this phenomenon occurs across combinations of different phosphotransferase (PTS) substrates with various gluconeogenic carbon sources and found it to depend on the transcription factor Mlc.

microbiology↗

bioRxiv2023

Long-term hematopoietic stem cells trigger quiescence in Leishmania parasites

Quiescence and posttreatment relapse constitute an important therapeutic constraint across the microbiological spectrum. This study unveils that Leishmania infantum and L. donovani parasites rapidly enter quiescence after an estimated 4-6 divisions in both mouse and human stem cells of the bone marrow but not in macrophages as primary host cells. Quiescent amastigotes display a reduced size and evidence for a rapid evolutionary adaptation response with genetic alterations. We formally demonstrate that acquisition of a quiescent phenotype endows parasites with a capacity to survive antileishmanial treatment. Transition through quiescence also results in an increased cellular infectivity and high transmission capacity through the sand fly vector. Transcriptional profiling of quiescent and non-quiescent metabolic states identified a limited set of 26 upregulated genes that are of particular interest given their predicted involvement as regulators of cell cycle progression and of gene expression at various levels. The differential gene set constitutes a reliable source for the identification of novel markers and potential drivers of quiescence, a metabolic state bestowing parasites the capacity to escape drug treatment.

microbiology↗

bioRxiv2023

Spatiotemporal development of growth and death zones in expanding bacterial colonies driven by emergent nutrient dynamics

Bacterial colony growth on hard agar is commonplace in microbiology; yet, what occurs inside a growing colony is complex even in the simplest cases. Robust colony expansion kinetics featuring a linear radial growth and a saturating vertical growth indicates a common developmental program which is elucidated here for Escherichia coli cells using a combination of modeling and experiments. Radial colony expansion is found to be limited by mechanical factors rather than nutrients as commonly assumed. In contrast, vertical expansion is limited by glucose depletion inside the colony, an effect compounded by reduced growth yield due to anaerobiosis. Carbon starvation in the colony interior results in substantial cell death within 1-2 days, with a distinct death zone that expands with the growing colony. Overall, the development of simple colonies lacking EPS production and differentiation is dictated by an interplay of mechanical constraints and emergent nutrient gradients arising from obligatory metabolic processes.

microbiology↗

bioRxiv2023

Modelling the impact of rainfall events on increasing Vibrio vulnificus concentrations in Pacific oysters (Crassostrea gigas)

Vibrio vulnificus occurs naturally in seawater and causes debilitating, often fatal illnesses, particularly in people with underlying health issues such as liver disease. The illness can occur when raw molluscan shellfish that have bio-accumulated the organism are consumed. New Zealand seafood is not known to have caused any illnesses, although there have been wound infections. This study sought to better understand the effect of environmental conditions on concentrations of V. vulnificus in Pacific oysters in three harbours over the summer months of 2016-19. Fortnightly sampling at two harbours only once gave a most probable number (MPN) of >10 per g of oyster meat, while the third harbour regularly produced much higher counts (up to 220,000 per g). From 2017-19, weekly samples from three sites in this harbour (four in 2018) were tested. Eleven peaks in concentration were observed, all when seawater temperatures exceeded 20{degrees}C and after heavy rainfall had reduced the seawater salinity, usually to <25{per thousand} from the average of 32{per thousand}. A fitted structural equation model with temperature and salinity terms accounted for 60% of the variance in concentrations and rates of decline after concentration peaks averaged 1.05 log10 MPN per week. The rate of decline was highly variable so microbiological testing would be required to confirm this rate for use in food safety management. However, the results of the study will enable better risk management. ImportanceVibrio vulnificus is a deadly bacterium that naturally occurs in some seawater and can be concentrated in shellfish such as oysters. Consumers with underlying health issues who eat contaminated shellfish may suffer serious illnesses. New Zealand shellfish have never been known to cause such illnesses although the species is present in our seawater. The strains present may not be able to cause foodborne illness. We found that V. vulnificus could be present in high concentrations in farmed Pacific oysters in one of three studied harbours. This happened when seawater temperatures were warm and heavy rainfall reduced salinity. We developed a model based on seawater temperature and salinity that would be able to predict concentrations in shellfish, at least in this harbour. This will help industry and regulators manage the food safety risk should this organism become a public health issue. TweetWe modelled concentrations of Vibrio vulnificus in Pacific oysters after flood events and showed that seawater salinity and temperatures in previous 168 h affected them.

microbiology↗

bioRxiv2023

Soil Prokaryotic and Fungal Biome Structures Associated with Crop Disease Status across the Japan Archipelago

Archaea, bacteria, and fungi in the soil are increasingly recognized as determinants of agricultural productivity and sustainability. A crucial step for exploring soil microbiomes with high ecosystem functions is to perform statistical analyses on potential relationship between microbiome structure and functions based on comparisons of hundreds or thousands of environmental samples collected across broad geographic ranges. In this study, we integrated agricultural field metadata with microbial community analyses by targeting > 2,000 soil samples collected along a latitudinal gradient from cool-temperate to subtropical regions in Japan (26.1- 42.8 {degrees}N). The data involving 632 archaeal, 26,868 bacterial, and 4,889 fungal operational taxonomic units detected across the fields of 19 crop plant species allowed us to conduct statistical analyses (permutational analyses of variance, generalized linear mixed models, and randomization analyses) on relationship among edaphic factors, microbiome compositions, and crop disease prevalence. We then examined whether the diverse microbes form species sets varying in potential ecological impacts on crop plants. A network analysis suggested that the observed prokaryotes and fungi were actually classified into several species sets (network modules), which differed substantially in associations with crop disease prevalence. Within the network of microbe-to-microbe coexistence, ecologically diverse microbes, such as an ammonium-oxidizing archaeum, an antibiotics-producing bacterium, and a potentially mycoparasitic fungus, were inferred to play key roles in shifts between crop-disease-promotive and crop-disease-suppressive states of soil microbiomes. The birds-eye view of soil microbiome structure will provide a basis for designing and managing agroecosystems with high disease-suppressive functions. IMPORTANCEUnderstanding how microbiome structure and functions are organized in soil ecosystems is one of the major challenges in both basic ecology and applied microbiology. Given the ongoing worldwide degradation of agroecosystems, building frameworks for exploring structural diversity and functional profiles of soil microbiomes is an essential task. Our study provides an overview of cropland microbiome states in light of potential crop-disease-suppressive functions. The large dataset allowed us to explore highly functional species sets that may be stably managed in agroecosystems. Furthermore, an analysis of network architecture highlighted species that are potentially used to cause shifts from disease-prevalent states of agroecosystems to disease-suppressive states. By extending the approach of comparative analyses towards broader geographic ranges and diverse agricultural practices, agroecosystem with maximized biological functions will be further explored.

microbiology↗

bioRxiv2023

A Far-Red Fluorescent Probe to Visualize Staphylococcus aureus in Patient Samples

Staphylococcus aureus (S. aureus) is the leading bacterial cause of death in high-income countries and can cause invasive infections at various body sites. These infections are associated with prolonged hospital stays, a large economic burden, considerable treatment failure, and mortality rates. So far, there is only limited knowledge about the specific locations where S. aureus resides in the human body during various infections. Hence, the visualization of S. aureus holds significant importance in microbiological research. Herein, we report the development and validation of a far-red-fluorescent probe to detect S. aureus in human biopsies from deep-seated infections. This probe displays strong fluorescence and low background in human tissues, outperforming current tools for S. aureus detection. Several applications are demonstrated, including fixed- and live-cell imaging, flow cytometry, and super-resolution bacterial imaging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/556223v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1bcf4e4org.highwire.dtl.DTLVardef@71bf45org.highwire.dtl.DTLVardef@1c468fdorg.highwire.dtl.DTLVardef@1c8baf3_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Experimental and methodological framework for the assessment of nucleic acids in airborne microorganisms

Studying airborne microorganisms is highly challenging due to ultra-low and spread biomass, and great spatial and temporal variabilities at short scales. Aeromicrobiology is still an emerging discipline of environmental microbiology, and some of the basic practices (replication, control of contaminants, etc) are not yet widely adopted, which potentially limits conclusions. Here we aim at evaluating the benefits of such practices in the study of the aeromicrobiome using molecular-based approaches, and recommend the following: (i) sample at high airflow rate, if possible into a fixative agent, in order to be able to capture specific situations ; (ii) replicate sampling and process samples individually to enable statistical analyses ; (iii) check for contaminants at different steps of the analytical process, and account for their potential stochasticity in sequence decontamination methods ; (iv) include internal references to verify qualitative and quantitative aspects of the data, and (v) eventually investigate multiple analytical procedures to identify potential impacts on the data. In our study, samples were collected at a remote mountain site using high-flow rate impingers collecting airborne material into nucleic acid preservation buffer. As high of [~]75% of the sequences were shared between independent triplicates, gathering 28 to 38% of the richness observed at the ASV level at a given sampling date, which also emphasizes spatial heterogeneity at short scale due to rare taxa. Thanks to replicates, daily variations in the diversity of bacteria could be distinguished statistically, and the inevitable presence of contaminating sequences in controls could be accounted for using established statistical methods. This work opens new perspectives and notably paves the way to untargeted molecular methods in the exploration of aeromicrobiomes composition and functioning. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/561683v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@48bddcorg.highwire.dtl.DTLVardef@1a8c3fforg.highwire.dtl.DTLVardef@dba98forg.highwire.dtl.DTLVardef@159055e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers.

Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics but their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using Escherichia coli DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown E. coli and Clostridium bolteae DSM 29485 to different doses of the toxin sodium arsenite. The growth of E. coli and C. bolteae was sensitive to arsenite doses of 1.3 M and 0.4 M, respectively. Complete inhibition of growth was achieved at 38 M arsenite for C. bolteae, and 338 M in E. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus. Importance statementWe present a workflow that facilitates the production and analysis of growth curves for anaerobic microbes using small-footprint microplate readers and an R script. This workflow is a cost and space-effective solution to most high-throughput solutions for collecting growth data from anaerobic microbes. This technology can be used for applications in which high-throughput would advance discovery, including microbial isolation, bioprospecting, co-culturing, host-microbe interactions, and drug/toxin-microbial interactions.

microbiology↗

bioRxiv2023

Feeding secondary fermentations with mammalian and fungal culture waste streams increases productivity and resource efficiency

The evolution of the circular bioeconomy will require the realisation of new value from waste streams generated from all manufacturing processes. Bioprocessing of recombinant proteins and functional foods using animal and microbial fermentations are fast growing industries with increasing volumes of spent culture media waste resulting in significant resource inefficiency. Here Chinese hamster ovary cell spent media (CSM) and Trametes versicolor fungal cell spent media (FSM) were used as model waste streams to feed Escherichia coli in secondary fermentations. Expression of a recombinant fusion protein was used to measure the value of these streams as feedstuff. E. coli cultures fed with either waste stream in bioreactors produced equivalent yields of the protein as those fed with rich microbiological media. Separately CSM was tested as a replacement feed for the fungal cell fermentation producing beta and alpha glucans. This waste-fed fermentation resulted in a 2-fold higher glucan yield than when using standard corn steep liquor feed. Quantitative analysis of the spent media content with mass spectrometry and hplc methods showed significant differences in elemental composition profiles, quantities of individual amino acids and carboxylic acids and levels of carbon sources. These findings underline the versatility of E.coli in utilisation of waste media as a feedstuff and show that the range of applications of CHO cell culture media waste are not limited to feeding bacterial fermentation. Exploration of cellular bioprocesses that can efficiently use cell media waste as fermentation feed will be a valuable step towards increased resource efficiency in bioprocessing industries. (249/250). Significance StatementThe expanding range and scale of valuable biomolecular products developed with bioprocessing activities using animal and microbial cells is responsible for the production of hundreds of millions of litres of waste culture media annually. Recycling this waste stream has the potential to decrease the impact of the associated high water and energy consumption contributing to meeting targets for the Paris 2050 Agreement and UN Sustainable Development Goals. This work shows that waste media from distinct bioprocesses can be reused to sustain high-level recombinant protein production in a secondary fermentation and the notable finding that a waste media can add value to a secondary process by increasing its productivity. Efficient valorisation of waste media is an urgent and scientifically valuable objective. (120/120)

microbiology↗

bioRxiv2023

Predicting Pseudomonas aeruginosa drug resistance using artificial intelligence and clinical MALDI-TOF mass spectra

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used in clinical microbiology laboratories for bacterial identification but its use for prediction of antimicrobial resistance (AMR) remains limited. Here, we used MALDI-TOF MS with artificial intelligence (AI) approaches to successfully predict AMR in Pseudomonas aeruginosa, a priority pathogen with complex AMR mechanisms. The highest performance was achieved for modern {beta}-lactam/{beta}-lactamase inhibitor drugs, namely ceftazidime/avibactam and ceftolozane/tazobactam, with area under the receiver operating characteristic curve (AUROC) of 0.86 and 0.87, respectively. As part of this work, we developed dynamic binning, a feature engineering technique that effectively reduces the high-dimensional feature set and has wide-ranging applicability to MALDI-TOF MS data. Compared to conventional methods, our approach yielded superior performance in 10 of 11 antimicrobials. Moreover, we showcase the efficacy of transfer learning in enhancing the performance for 7 of 11 antimicrobials. By assessing the contribution of features to the models prediction, we identified proteins that may contribute to AMR mechanisms. Our findings demonstrate the potential of combining AI with MALDI-TOF MS as a rapid AMR diagnostic tool for Pseudomonas aeruginosa.

microbiology↗

bioRxiv2023

What is Slough?A pilot study to define the proteomic and microbial composition of wound slough and its implications for wound healing.

Slough is a well-known feature of non-healing wounds. This study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Twenty-three subjects with slow-to-heal wounds and visible slough were enrolled. Etiologies included venous stasis ulcers, post-surgical site infections, and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analyzed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound etiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation, and immune processes. To predict variables associated with wound healing, protein, microbial, and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterize wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilized biomarker.

microbiology↗

bioRxiv2023

The microbial community of rust layer biofilm was driven by seawater microbial community

Microbiologically influenced corrosion (MIC) accounts for approximately 20% of the total corrosion-related losses worldwide, causing significant economic damage each year, particularly in Marine environments. However, there are still no truly effective and eco-friendly protection solutions against MIC, among which the incomplete understanding of the microbial biofilm development on metallic surface is a key limitation. Using 16S rRNA and ITS sequencing, we studied bacterial and fungal communities in rust layer biofilm and seawater. The results showed that Proteobacteria, Cyanobacteria and Bacteroidota were the dominant bacterial phyla, and Ascomycota and Basidiomycota were the dominant fungal phyla both in the rust layer biofilm and seawater. Bacteria like Erythrobacter and Aquimarina, as well as fungi like Aspergillus and Acremonium were were notable microorganisms in the rust layer biofilm. Source analysis revealed differences between biofilm and seawater communities, with 23.08% bacterial and 21.48% fungal communities originating from seawater. Stochastic processes governed biofilm and seawater microbial communities, and network analysis showed coexistence and interaction among bacteria and fungi. IMPORTANCEThe composition and source analysis of bacterial and fungal communities in the rust layer and seawater were studied, and the interaction of bacteria and fungi in the rust layer was discussed for the first time. Based on these findings, we provided a framework to explain the observed characteristics of microbial communities in rust layer biofilm and presented key evidence supporting the relationship between different microbial structures and interactions with metal corrosion. These findings, from the perspective of microbial ecology, provide a theoretical foundation for studying microbial corrosion in marine environments.

microbiology↗