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bioRxiv2021

Methylothon: a versatile course-based high school research experience in microbiology and bioinformatics-- with pink bacteria

Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here we present an overview of the modules learning goals, describe course resources (available for public use on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from Spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees, and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

scientific communication and education↗

bioRxiv2022

Use of Fe3O4 nanoparticles in reactor co-digestion of residues from 1G2G ethanol biorefinery: microbiological routes and operational aspects

The co-digestion of residues from the sugarcane industry has already proven to be a highly attractive process for biogas production through anaerobic digestion (AD). The use of residues such as vinasse (1G) filter cake (1G) and deacetylation liquor (2G) in CSTR operation showed the potential for integrating 1G and 2G ethanol biorefineries through AD in previous work by our research group. The use of nanoparticles (NP) is a favorable way to optimize AD processes, as these additives allow the introduction of nutrients to the process more assertively concerning the distribution and interaction with microorganisms. The present work proposed the optimization of the co-digestion of vinasse, filter cake, and deacetylation liquor in a continuous reactor by adding Fe3O4 NP, comparing the results with a previous reactor operation without NP. Initially, tests were carried out in batches with different NP concentrations, resulting in 5 mg L-1 as the best concentration to be added in the continuous reactor along the increments of the applied organic rate load (ORL). CH4 production reached a maximum value of 2.8 {+/-} 0.1 NLCH4 gVS-1 and the organic matter removal reached 71 {+/-} 0.9%, in phase VI (ORL of 5.5 gVS L-1 day-1). This production was 90% higher than the reactor co-digestion operation without NP. The pH and alkalinity results indicated the methanogenesis stabilization within 60 days of operation: 30 days before when there was no NP added. The AD development was stable, presenting low variations in the oxidation-reduction potential (ORP) and stable organic acid (OA) concentrations, which indicated the propionic acid route to produce CH4 was predominant. The main methanogenic Archeae identified was Methanoculleus, indicating that the predominant metabolic route was that of acetate (SAO) coupled with hydrogenotrophic methanogenesis. The use of Fe3O4 NP managed to improve the AD from the 1G2G ethanol production residues and stimulated the microbial community growth, not modifying the preferable metabolic pathways.

bioengineering↗

bioRxiv2022

Fragment size and diversity of mulches affect their decomposition, nutrient dynamics, and soil microbiology

Plant-based mulch has been proposed as a sustainable way of maintaining soil fertility. However, the role of mulch diversity, quality, and size in decomposition dynamics, and their effect on crop yield are not fully explored. We investigated how mulch quality, proxied by the constituent plant species diversity, and residue size drive mulch decomposition, crop nutrition, and yield. A rhizotron experiment was set up with barley as a model crop, with the addition of mulch of two particle sizes (1.5 and 30 cm) and four different plant residue mixes (17, 12, 6, and 1 species) in a fully factorial design. Soil nutrient dynamics were measured at advanced decomposition stages, together with residue quality, arbuscular mycorrhizal fungal (AMF) root colonization, and crop yield. Residue mass loss was significantly affected by its chemical composition. Long residues retained significantly higher C and N content, than short residues. Crop yield was not affected by residue type or size. Residue size significantly affected barley growth rate, influencing seed protein content. Soil available K was significantly increased by residues with a higher initial C:N ratio. Short residues resulted in higher soil Zn. Residues of higher diversity resulted in higher AMF root colonization of the barley plants. Generally, long residue mulches maintain soil fertility for a longer period than short ones, without a deleterious effect on crop yield. Further investigation should evaluate the effect of continuous application of long residue mulches on soil fertility and microbial populations.

ecology↗

bioRxiv2022

Efficient Multi-Label Attribute Classification and Recognition of Microbiological Bacteria Based on Deep Learning and model fine-tuning

Bacterial vaginosis (BV) is the most common gynecological complaint affecting health of a large percentage of women worldwide. Traditional manual microscopy methods are expensive and time-consuming, to improve accuracy and efficiency, automated bacterial identification devices with detection intelligence algorithms are urgently needed. We propose a Fine-tuned SmallerVGG (FTS-VGG) deep convolutional network model based multi-label classification method for bacteria. Comparison experiments were deployed on several advanced backbone networks, including transfer learning on pre-trained VGG19, demonstrating that the proposed method achieves the advantages of being lighter, faster, more accurate and more efficient. Due to the high cost of time and expertise of experienced clinicians, we use random erasing for data augmentation to address the challenge of dataset collection and annotation, experiments demonstrate its robustness to occlusion. The proposed method has theoretical and practical implications, as well as the potential to be widely extended to other microscopic imaging applications.

biochemistry↗

bioRxiv2023

Reduction, removal or replacement of sodium nitrite in a model of cured and cooked meat: a joint evaluation of consequences on microbiological issues in food safety, colon ecosystem and colorectal carcinogenesis

ScopeEpidemiological and experimental evidence reported that processed meat consumption is associated with colorectal cancer (CRC) risk. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Methods and resultsCompared to the reference level (120 mg/kg of ham), the effects of sodium nitrite reduction (90 mg/kg of ham), removal and replacement were analysed on ham characteristics and in a CRC rat model. Sodium nitrite removal and reduction induced a similar decrease in CRC preneoplastic lesions, but only reduction led to (i) an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level of 120 mg/kg and (ii) an effective control of lipid peroxidation. Among the three alternatives tested, none led to a significant gain when compared to the 120 mg/kg ham reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions but the concomitant presence of nitrosyl iron in feces. Except vegetable stock, other alternatives were less efficient than sodium nitrite ([≥] 90 mg/kg) in reducing L. monocytogenes growth. ConclusionNitrite reduction (90mg/kg) effectively reduced CRC risk through limiting NOC formation and lipid peroxidation, while mitigating L. monocytogenes risks from cooked hams. Going further in reduction should be possible if accompanied by antioxidants to limit lipid peroxidation and appropriate use-by dates.

pathology↗

bioRxiv2024

Computational microbiology of soil organic matter mineralization: Use of the concept of curve skeleton to partition the 3D pore space in computed tomography images

Recent advances in 3D X-ray Computed Tomography (CT) sensors have stimulated research efforts to unveil the extremely complex micro-scale processes that control the activity of soil microorganisms. Classical methods for the numerical simulation of biological dynamics using meshes of voxels, such as the Lattice Boltzmann Method (LBM), tend to require long computation times. The use of more compact geometrical representations of the pore space can drastically decrease the computational cost of simulations. Recent research has introduced basic analytic volume primitives to define piece-wise approximations of the pore space to simulate drainage, diffusion, and microbial mineralization of organic matter in soils. Such approaches work well but a drawback is that they give rise to non-negligible approximation errors. In the present article, another alternative is proposed, where pore space is described by means of geometrically relevant connected subsets of voxels (regions) regrouped on the basis of the curve linear skeleton (3D medial axis). This curve skeleton has been adopted to characterize 3D shapes in various fields (e.g., medical imaging, material sciences, etc.) but the few publications that have used it in the context of soils, have dealt exclusively with the determination of pore throats. This technique is used mostly to describe shape and not to partition it into connected subsets. Here, the pore space is partitioned by using the branches of the curve skeleton, then an attributed relational graph is created in order to simulate numerically the microbial mineralization of organic matter, including the diffusion of by-products. This new representation can be used for graph-based simulations, which are different from voxel-based simulations.

bioinformatics↗

bioRxiv2025

Evaluation of Antioxidant Anticancer and Antimicrobial Activities of Datura Leaf Extracts: A Microbiological Perspective

Datura, a genus of flowering plants, has long been used in traditional medicine owing to its pharmacological properties. This study investigated the antioxidant and antimicrobial activities of Datura leaf extracts by using in vitro assays. Antioxidant potential was evaluated using standard methods, while antimicrobial activity was assessed against various bacterial and fungal strains using the disk diffusion method. The results demonstrated significant antioxidant properties and broad-spectrum antimicrobial activity, supporting the potential of Datura as a source of bioactive compounds for pharmaceutical applications. The findings of this study elucidate the potential of datura leaf extracts as natural alternatives to synthetic antioxidants and antimicrobial agents in various industries. Further research is warranted to explore the specific bioactive compounds responsible for these properties and to investigate their mechanisms of action. Additionally, future studies should focus on optimizing extraction methods to enhance the potency and efficacy of Datura-derived products for potential therapeutic applications.

bioengineering↗

bioRxiv2025

Disentangling the importance of microbiological and physicochemical properties of Ethiopian field soils for the Striga seed bank and sorghum infestations

Striga hermonthica (Striga) is a parasitic weed that severely affects sorghum yields in sub-Saharan Africa. Recent studies highlighted the soil microbiomes potential to suppress Striga through interference with specific stages in its life cycle. In this study, meta-analysis of 48 Ethiopian field soils revealed that microbial communities and their interactions with soil physico-chemical properties correlated with Striga field occurrence. Striga infestation of sorghum and soil seedbank levels were negatively correlated with clay content and the nutrients potassium, sulfur, calcium, and carbon. Microbiome analyses indicated that fungal communities were more responsive than bacteria to changes in Striga infestation and seedbank levels, with distinct microbial compositions even in soils where Striga was not detected. Specific fungal and bacterial genera showed both positive and negative correlations with Striga measures, but patterns rarely held across taxonomic levels, highlighting the complexity of microbiome-Striga interactions. To begin to validate these correlations, we tested an isolate from the fungal genus Neocosmospora, which negatively correlated with the Striga seedbank, and showed that this isolate promotes Striga seed germination in vitro, suggesting potential for biological control of Striga. The data and analysis methods are integrated and shared in a public Shiny App for broader analysis and continued research on soil-Striga interactions.

plant biology↗

bioRxiv2024

microGalaxy: A gateway to tools, workflows, and training for reproducible and FAIR analysis of microbial data

The explosion of microbial omics data has outpaced the ability of many researchers to analyze it, with complex tools and limited computational resources creating barriers to discovery. To address this gap, we present the Microbiology Galaxy Lab: a free, globally accessible, community-supported platform that combines state-of-the-art analytical power with user-friendly accessibility. Supported by the Galaxy and global microbiology communities, this platform integrates over 315 tool suites and 115 curated workflows, enabling comprehensive metabarcoding, (meta)genomic, (meta)transcriptomic, and (meta)proteomic data analysis within a FAIR-aligned environment. It also supports research in the health and infectious disease sectors, as well as in environmental microbiology. The platforms utility is exemplified through various use cases, including antimicrobial resistance tracking, biomarker prediction, microbiome classification, and functional annotation of key microbes. Built on reproducibility and community engagement, it supports creation, sharing, and updating of best-practice workflows. Over 35 tutorials and learning paths empower scientists, fostering an ecosystem that keeps resources at the forefront of microbial science. The Microbiology Galaxy Lab enables collective analysis, democratising research, thereby accelerating discovery across the global microbiology community (microbiology.usegalaxy.org, microbiology.usegalaxy.eu, microbiology.usegalaxy.org.au, microbiology.usegalaxy.fr).

bioinformatics↗

bioRxiv2021

Evaluation of flow cytometry for cell count and detection of bacteria in biological fluids.

The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The gold standard method for cell count in biological fluids is the manual method using counting chambers. The microbiological routine procedures consist of Direct Gram staining and culture on solid or liquid media. We evaluate the analytical performance of SYSMEX UF4000 (Sysmex, Kobe, Japan) and Sysmex XN10 (Sysmex, Kobe, Japan) in comparison with cytological and microbiological routine procedures. A total of 526 biological fluid samples were included in this study (42 ascitic, 31 pleural, 31 peritoneal, 125 cerebrospinal, 281 synovial, and 16 peritoneal dialysis fluids). All samples were analysed by flow cytometry and subsequently processed following cytological and/or microbiological routine procedures. With regards to cell counts, UF4000 (Sysmex, Kobe, Japan) showed a performance which was at least equivalent to those of the reference methods and superior to those of XN10 (Sysmex, Kobe, Japan). Moreover, the bacterial count obtained with UF4000 (Sysmex, Kobe, Japan) was significantly higher among culture or Direct Gram stain positive samples. We established 3 optimal cut-off points to predict Direct Gram stain positive samples for peritoneal (465.0 bacteria/L), synovial (1200.0 bacteria/L), and cerebrospinal fluids (17.2 bacteria/L) with maximum sensitivity and negative predictive values. Cell count and detection of bacteria by flow cytometry could be used upstream cytological and microbiological routine procedures to improve and accelerate the diagnosis of infection of biological fluid samples.

microbiology↗

bioRxiv2024

Vaginal microbiota as a predictor of preterm birth: an observational cohort study

RationaleIncreasing evidence links vaginal microbiota composition and preterm birth (PTB). However, most metagenomic studies are relatively small-sized, do not systematically adjust for confounders, and are difficult to transpose in clinical settings. ObjectiveTo identify, using routine vaginal microbiological cultures, signatures of preterm labor (PTL), preterm premature rupture of membranes (PPROM), and PTB. MethodsWe conducted an observational cohort study from August 2018 until June 2023 in France. Pregnant women were enrolled in three groups: control, PTL and PPROM. Demographic, clinical data, and pregnancy outcome were collected. Vaginal swabs were collected at enrollment and microbiological cultures were performed. The association between bacterial species and PTL, PPROM, and PTB was studied in univariate analyses. Adjusted odds ratio (aOR) and 95% confidence intervals (CI) were calculated in multivariable analyses adjusting for confounding variables. Results1,848 women were included: 1,048 in the control group, 417 with PTL, and 383 with PPROM. Among women with PTL or PPROM, 328/646 (50.8%) spontaneously delivered preterm. Vaginal samples enriched in enterobacteria and in Gardnerella spp. were signatures of PTL. Lactobacilli depletion and enterobacteria enrichment were signatures of PPROM. In multivariable analysis, lactobacilli depletion was the strongest risk factor for spontaneous PTB (aOR 2.29, 95% CI 1.52-3.48). ConclusionsOur study corroborates previous findings demonstrating the protective role of lactobacilli during pregnancy and highlights enterobacteria as signatures of PTL and PPROM. Furthermore, it provides perspectives for the management of women at high risk of PTB using standard microbiological techniques. HighlightsO_LIVaginal microbiological analysis of 800 women with preterm labor or PPROM C_LIO_LIPreterm labor signatures include enterobacteria and Gardnerella enrichment C_LIO_LIPPROM signatures include lactobacilli depletion and enterobacteria enrichment C_LIO_LILactobacilli depletion is the strongest risk factor for preterm birth C_LI

microbiology↗

bioRxiv2019

Original research. Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts ({Delta}Cp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0 %), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youdens index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

microbiology↗

bioRxiv2026

Microbial biodeterioration of eighteenth-century oil paintings in Orosi, Costa Rica, and in vitro evaluation of volatile essential oil components as antimicrobials

Microbial colonization is a major cause of deterioration in paintings, leading to discoloration, pigment degradation, and loss of structural integrity. While biodeterioration of artworks has been studied in temperate climates, tropical environments remain underexplored despite their high humidity and temperature, which promote microbial growth. This study assessed the microbiological deterioration of two eighteenth-century oil paintings, La Muerte de San Jose and Virgen de Guadalupe, located in Orosis Colonial Church and Religious Art Museum, Costa Rica. Microorganisms were isolated and identified using VITEK(R) 2, microscopy, and MALDI-ToF analysis, and their biofilm-forming capacity was evaluated. Additionally, the antimicrobial activity of six essential oil components was tested using direct and indirect contact assays. Twenty-three bacterial species and fifteen fungal genera were identified, with Bacillus, Staphylococcus, Cladosporium, and Aspergillus among the most common. Notably, La Virgen de Guadalupe displayed the highest microbial diversity, reflected in a high Shannon index, indicative of a more complex microbial community. Several isolates displayed strong biofilm formation, particularly Bacillus subtilis/amyloliquefaciens/vallismortis and Staphylococcus saprophyticus. Linalool exhibited the strongest inhibitory activity, achieving complete bacterial growth inhibition in non-contact assays. Environmental monitoring revealed persistently elevated relative humidity and CO2 levels during the study period. Together, these results reveal the complex microbial ecology of tropical heritage paintings and demonstrate that volatile essential oil components can serve as candidates for low-impact antimicrobial strategies in preventive conservation. ImportanceUnderstanding the microbiological deterioration of cultural heritage in tropical environments is crucial for designing sustainable conservation strategies. While microbial colonization of artworks has been widely studied in temperate regions, data from tropical climates remain limited despite inherently favorable conditions for microbial proliferation. This study integrates microbiological, environmental, and physicochemical analyses to characterize microbial communities colonizing eighteenth-century oil paintings in Orosi, Costa Rica. By combining microbial identification, biofilm quantification, and essential oil biocide testing, it bridges applied microbiology and cultural heritage conservation. The finding that volatile components such as linalool inhibit biofilm-forming bacteria without direct contact highlights their potential as eco-friendly, noninvasive antimicrobial alternatives to conventional biocides. These results expand the understanding of biodeterioration dynamics under tropical conditions and offer a practical framework for developing sustainable, evidence-based conservation protocols that protect both heritage materials and the environment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/723565v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@16cd608org.highwire.dtl.DTLVardef@57aa00org.highwire.dtl.DTLVardef@159fcbeorg.highwire.dtl.DTLVardef@e0363b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0.C_FLOATNO Artistic visualization of the geographical context of the studied artworks and the multidisciplinary analytical approaches applied, highlighting the diversity of microorganisms identified (illustration by Keylin Urena-Alvarado). C_FIG

microbiology↗

bioRxiv2020

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics.

Antibiotics are failing fast, and the development pipeline is alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which are inhibitors of bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is often stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors. Inhibitors of DsbA block bacterial oxidative protein folding and were previously identified by biophysical and biochemical assays. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that a combination of a cell-based AssT sulfotransferase assay and the UPEC motility assay, modified for a higher throughput format, can provide a robust and target-specific platform for the evaluation of DsbA inhibitors. Our pipeline could also be used in fragment and compound screening for the identification of new DsbA inhibitor classes or hits with a broad spectrum of activity. In conclusion, the establishment of accurate, high-throughput microbiological assays for antivirulence drug identification and early preclinical development, is a significant first step towards their translation into effective therapeutics. ImportanceThe safety net of last resort antibiotics is quickly vanishing as bacteria become increasingly resistant to most available drugs. If no action is taken, we will likely enter a post-antibiotic era, where common infections and minor injuries are once again lethal. The paucity in new antibiotic discovery of the past decades has compounded the problem of increasing antibiotic resistance, to the point that it now constitutes a global health crisis that demands global action. There is currently an urgent need for new antibacterial drugs with new targets and modes of action. To address this, research and development efforts into antivirulence drugs, such as DsbA inhibitors, have been ramping up globally. However, the development of microbiological assays as tools for effectively identifying and evaluating antivirulence drugs is lagging behind. Here, we present a high-throughput cell-based screening and evaluation pipeline, which could significantly advance development of DsbA inhibitor as antivirulence therapeutics.

microbiology↗

bioRxiv2021

Assessing antibiotic tolerance of Staphylococcus aureus derived directly from patients by the Replica Plating Tolerance Isolation System- REPTIS

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required. We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics. One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation. Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates. In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.

microbiology↗

bioRxiv2021

Locating and Quantifying Carbon Steel Corrosion Rates Linked to Fungal B20 Biodiesel Degradation

Fungi that degrade B20 biodiesel in storage tanks have also been linked to microbiologically influenced corrosion (MIC). A member of the filamentous fungal genus Byssochlamys, and a yeast from the genus Wickerhamomyces were isolated from heavily contaminated B20 storage tanks from multiple Air Force bases. Although these taxa were linked to microbiologically influenced corrosion in situ, precise measurement of their corrosion rates and pitting severity on carbon steel was not available. In the experiments described here, we directly link fungal growth on B20 biodiesel to higher corrosion rates and pitting corrosion of carbon steel under controlled conditions. When these fungi were growing solely on B20 biodiesel for carbon and energy, consumption of FAME and n-alkanes was observed. The corrosion rates for both fungi were highest at the interface between the B20 biodiesel and the aqueous medium, where they acidified the medium and produced deeper pits than abiotic controls. Byssochlamys produced the most corrosion of carbon steel and produced the greatest pitting damage. This study characterizes and quantifies the corrosion of carbon steel by fungi that are common in fouled B20 biodiesel through their metabolism of the fuel, providing valuable insight for assessing MIC associated with storage and dispensing B20 biodiesel. IMPORTANCEBiodiesel is widely used across the United States and worldwide, blended with ultralow sulfur diesel in various concentrations. In this study we were able to demonstrate that the filamentous fungi Byssochlamys AF004 and the yeast Wickerhamomyces SE3 were able to degrade fatty acid methyl esters and alkanes in biodiesel causing increases in acidity. Both fungi also accelerated the corrosion of carbon steel, especially at the interface of the fuel and water, where their biofilms were located. This research provides controlled, quantified measurements and the localization of microbiologically influenced corrosion caused by common fungal contaminants in biodiesel fuels.

microbiology↗

bioRxiv2022

Reducing High Pressure Processing Costs: Efficacious Alternatives to Current Standard Procedures in the Food Manufacturing Industry

As a result of recent advancements in design and optimization of high-pressure processing units, the technology is gaining rapid adoption across various sectors of food manufacturing, thus requiring extensive microbiological hurdle validation studies for efficacious and feasible utilization of the technology. Commercial adoption of high-pressure processing is gaining momentum of industrial importance because of recent advances in the engineering of pressure-based pasteurization units. With tremendous ability of plethora of microorganisms to move towards fitness through vertical and horizontal gene transfer mechanisms, prevention of natural and anthropogenic pathogens of public health concern is a daunting task and a moving target. Current study discusses, Reducing the Cost Associated with High Pressure Processing: Efficacious Alternatives to the Current Standard Procedure in the Food Manufacturing Industry, with microbiological challenge studies for inactivation of the pathogen exposed to various times and intensity levels of elevated hydrostatic pressure (Pressure BioScience Inc.). Elevated hydrostatic pressure is a non-thermal procedure that exposes pathogens to pressures of up to 80,000 PSI (>550 MPa). Various times (3, 4, and 5 minutes) at pressure intensity levels of 600 MPa, (87K PSI), 550 MPa (79K PSI), 480 MPa (70K PSI), 415 MPa (60K PSI), and 345 MPa (50K PSI) of elevated hydrostatic pressure (Hub880 Explorer, Pressure BioScience Inc), were investigated at 4{degrees}C and for 45{degrees}C for inactivation of Shiga toxin-producing Escherichia coli O157:H7 (STEC) (ATCC numbers BAA 460, 43888, 43894, 35150, 43889, and 43890) respectively, Big Six non-O157 Shiga toxin-producing E. coli (nSTEC) (ATCC numbers BAA 2196, 2193, 2215, 2440, 2219, and 2192) respectively, Salmonella serovars (ATCC numbers 13076, 8387, 6962, 9270, and 14028) respectively, and Listeria monocytogenes (ATCC numbers 51772, 51779, BAA 2657, and BAA 13932). Studies were conducted in two biologically independent repetitions as a blocking factors of a randomized complete block design containing three repetitions per time/treatment within each block, analyzed statistically using GLM procedures of SAS 9.4 software at type one error level at 5% using Tukey- and Dunnett-adjusted ANOVA. A Barocycler Hub840 unit (Pressure BioScience Inc., Southeastern, MA), equipped with a water jacket and circulating water bath for precise application of hydrostatic pressure and controlled temperature was utilized. Up to 0.95 and 2.60 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli Non-O157 at planktonic stages were achieved using application of pressure at 345 MPa and 550 MPa for 5 minutes and 4 minutes, respectively at 4{degrees}C. Up to 4.42 and 5.10 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli Non-O157 at planktonic stages were achieved using application of pressure at 345 MPa and 480 MPa for 5 minutes and 4 minutes, respectively at 45{degrees}C. Up to 1.63 and 3.14 log reductions (P < 0.05) of non-habituated Listeria monocytogenes at planktonic stages were achieved using application of pressure at 345 MPa and 600 MPa for 5 minutes and 3 minutes, respectively at 4{degrees}C. Up to 4.91 and 6.37 log reductions (P < 0.05) of non-habituated Listeria monocytogenes at planktonic stages were achieved using application of pressure at 550 MPa and 480 MPa for 4 minutes, respectively at 45{degrees}C. Up to 2.87 and 5.82 log reductions (P< 0.05) of non-habituated Salmonella serovars at planktonic stages were achieved using application of pressure at 345 MPa and 550 MPa for 5 minutes and 4 minutes, respectively at 4{degrees}C. Up to 5.17 and 6.79 log reductions (P< 0.05) of non-habituated Salmonella serovars at planktonic stages were achieved using application of pressure at 415 MPa and 600 MPa for 5 minutes and 3 minutes, respectively at 45{degrees}C. Up to 0.86 and 1.35 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli O157:H7 at planktonic stages were achieved using application of pressure at 345 MPa and 480 MPa for 5 minutes and 4 minutes, respectively at 4{degrees}C. Up to 2.02 and 6.12 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli O157:H7 at planktonic stages were achieved using application of pressure at 345 MPa and 550 MPa for 5 minutes and 4 minutes, respectively at 45{degrees}C. Results of this study could be incorporated as a part of predictive public health microbiology modeling and risk assessment analysis for prevention of pathogen related disease and illness episodes.

microbiology↗

bioRxiv2023

Indigenous Reduction of Multi-drug Resistant Bacterial Pathogens in a Natural Uncontrolled Fermented Milk Ecosystem

Raw milk and its products have been questioned for microbiological safety. However, Dahi: an artisanally fermented milk product that is yet microbiologically unexplored is popularly known for therapeutic uses in public. Aiming safety and therapeutic assessment of dahi, samples of raw milk and dahi were analysed for microbiology and bacterial pathogens. The pathogens were also evaluated for antibiotic resistance. Further dahi samples were assessed for antipathogen effect. The total bacterial count of raw milk was between 3.80 x 1002 to 2.97 x 1007 and total fungal count was 2.50 x 1001 to 4.90 x 1002 whereas total bacterial and fungal count of Dahi was 3.00 x 1001 to 4.05 x 1009 and 2.00 x 1001 to 5.30 x 1007. Bacterial population of the Dahi was found dominated by lactic acid bacteria. In milk samples, E. coli was detected as 38%, S. aureus 28%, L. monocytogenes 3%, Salmonella 42% and Pseudomonas spp 31%. In Dahi samples, their percentages were 7%, 10%, 7%, 8% and 2% respectively. The detected pathogens were found resistant against different antibiotics especially to third and fourth generations of cephalosporin and other beta-lactam drugs. When assessed invitro, the low incidence of pathogens in the Dahi samples were associated to its inhibitory effect against pathogens. Dahi samples also inhibited the growth of antibiotic resistance ATCC strains. The inhibitory activity of the Dahi is due to the changes occurs during fermentation but not corelated to the pH of the Dahi.

microbiology↗