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bioRxiv2021

Molecular Epidemiology and Microbiological Characteristics of Cryptococcus gattii VGII isolates from China

Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, and combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from china exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors (temperature, melanin production, and capsule size) and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China. Author SummaryCryptococcus gattii is one of the life-threatening fungal pathogens that can infect immunocompetent individuals. C.gattii can be invaded through the respiratory tract and can be spread to the brain, causing meningoencephalitis and even death. In 1999, an outbreak of C. gattii VGII occurred on Vancouver Island in Canada and spread in the Pacific Northwest region, which attracted more attention. Compared with other nations, the data of C gattii VGII in China was limited. To better understand the molecular epidemiology and microbiological characteristics of C.gattii VGII isolates from China, Bioinformatics analysis and in vivo and in vitro experiments were performed for clinical strains. Multi-locus Sequence Typing and whole-genome sequencing reveal the genetic diversity of the isolates and genetic relationships with isolates from other countries. We also investigated the antifungal drug susceptibility and virulence differences of the isolates. The authors expect that our work can provide useful data for the prevention and control of C. gattii VGII in China and to draw attention to C. gattii.

microbiology↗

bioRxiv2023

High Throughput Viability Assay for Microbiology

Counting viable cells is a universal practice in microbiology. The colony forming unit (CFU) assay has remained the gold standard to measure viability across disciplines; however, it is time-intensive and resource-consuming. Herein, we describe the Geometric Viability Assay (GVA) that replicates CFU measurements over 6-orders of magnitude while reducing over 10-fold the time and consumables. GVA computes a samples viable cell count based on the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with gram-positive and -negative planktonic bacteria, biofilms, and yeast. Laborious CFU experiments such as checkerboard assays, treatment time-courses, and drug screens against slow-growing cells are simplified by GVA. We therefore screened a drug library against exponential and stationary phase E. coli leading to the discovery of the ROS-mediated, bactericidal mechanism of diphenyliodonium. The ease and low cost of GVA evinces it can accelerate existing viability assays and enable measurements at previously impractical scales.

microbiology↗

bioRxiv2024

Microbiology and Anthropology: a multidisciplinary approach for estimating time since death during Winter in rural New South Wales, Australia.

Accurate determination of the post-mortem interval (PMI) is critical in forensic casework. Most studies conclude that PMI determinations are dependent on local climatic and geographical factors. Despite this, there is little data in an Australian environment outside of entomological studies. In the absence of insect data on or around the remains, alternative methods are required. Anthropological methods of observing and scoring the extent of decomposition at the time of discovery provide only broad estimates of PMI. Microbial succession is responsible for these observable soft tissue changes, particularly in early and late decomposition. The aim of this study was to combine anthropological and microbiological methods to provide data for determining PMI in a temperate Australian climate. Microbial DNA was isolated from skin and cavity swabs and used to perform 16S rRNA metagenomic analysis of pooled DNA samples to allow the choice of target taxa for qPCR. qPCR indicated significant changes in microbial communities with a dominant population of Gammaproteobacteria at early time points giving way to Firmicutes and Bacteroidetes near the end of the experiment. Expressing qPCR data as a ratio of Lactobacillales/Enterobacteriaceae provided data that could be useful in determining early and late decomposition. The genus Psychrobacter was identified as a good indicator of late decomposition in winter conditions. qPCR analysis, with further refinements, could be part of an accurate quantitative method of determining PMI.

microbiology↗

bioRxiv2025

Towards Clinical Phage Microbiology for Biofilm Infections: A Comparative Study and a Suggested Protocol

Non-resolving bacterial infections involve biofilm formation and are often complicating treatments. Utilizing lytic bacteriophages with antibiotics holds promise in biofilm eradication. Accurately matching phage-antibiotic combinations against target bacteria, termed Clinical Phage Microbiology (CPM), is crucial in effective phage therapy treatments. However, compared to planktonic cultures, performing CPM on biofilm infections poses a significant challenge due to the lack of effective methods and a standard protocol. This study compared various CPM approaches in biofilms. To this end, the activity of five phages was assessed against Pseudomonas aeruginosa biofilms using nine methods and multiple approaches. Here, we discuss various aspects of each technique, including sensitivity, duration, ease of implementation in diagnostic labs, and labor. Finally, we offer a preliminary protocol for testing phage-sensitivity in biofilm, which was tested on various bacteria species, and may serve as a basis for comprehensive CPM in biofilm.

microbiology↗

bioRxiv2018

Nanopore-based DNA sequencing in clinical microbiology: preliminary assessment of basic requirements

AimIdentify basic requirements for a metagenomic nanopore sequencing protocol permitting frequent application in a clinical microbiology daily routine diagnostic setting.\n\nBackgroundNanopore sequencing with the Oxford Nanopore Technologies MinION device has a potential to markedly improve clinical diagnosis of infections. Reports have emerged recently that it may provide direct-from-clinical-sample information; for example, with urine samples, bronchial tuberculosis samples and orthopedic prostheses. However, the ideal protocol for clinical use remains to be determined, especially relating to detection of relevant pathogen quantities and to finding a reasonable level of economic costs.\n\nResultsMinION can provide qualitatively and quantitatively correct identification of multiple species in metagenomics samples. For detection of clinically relevant quantities of bacteria (on a nanogram DNA level) there is a need for carrier DNA. Importantly, high-purity DNA and a naive MinION flow cell seem to be critical parameters.\n\nConclusionsOur results suggest that high-purity clinical sample DNA, addition of carrier DNA and a naive flow cell are critical factors for clinical use of MinION. A relatively high error rate may limit detection of antimicrobial resistance genes, and a realistic level of costs will require availability of a price-reduced and single-use flowcell.

microbiology↗

bioRxiv2018

Characterization of emetic and diarrheal Bacillus cereus strains from a 2016 foodborne outbreak using whole-genome sequencing: addressing the microbiological, epidemiological, and bioinformatic challenges

The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) has not been routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group strains were obtained from human cases (n =7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (clade III) and B. cereus sensu stricto (clade IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely-related and formed a well-supported clade relative to publicly-available emetic clade III genomes with an identical sequence type (ST 26). When compared to publicly-available emetic clade III ST 26 B. cereus group genomes, the 30 emetic clade III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly-available genomes by a mean of 301.7 to 528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. Based on both WGS and epidemiological data, we hypothesize that the outbreak was a single source outbreak caused by emetic clade III B. cereus belonging to the B. paranthracis species. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

microbiology↗

bioRxiv2018

Linking Fungal and Bacterial Proliferation to Microbiologically Influenced Corrosion in B20 Biodiesel Storage Tanks

Biodiesel is a renewable substitute, or extender, for petroleum diesel that is composed of a mixture of fatty acid methyl esters (FAME) derived from plant and animal fats. Ultra-low sulfur diesel (ULSD) blended with up to 20% FAME can be used interchangeably with ULSD, is compatible with existing infrastructure, but is also more susceptible to biodegradation. Microbial proliferation and fuel degradation in biodiesel blends has not been directly linked in situ to microbiologically influenced corrosion. We, therefore, conducted a yearlong study of B20 storage tanks in operation at two locations, identified the microorganisms responsible for observed fuel fouling and degradation, and measured in situ corrosion. The bacterial populations were more diverse than the fungal populations, and largely unique to each location. The bacterial populations included members of the Acetobacteraceae, Clostridiaceae, and Proteobacteria. The abundant Eukaryotes at both locations consisted of the same taxa, including a filamentous fungus within the family Trichocomaceae, and the Saccharomycetaceae family of yeasts. Increases in the absolute and relative abundances of the Trichocomaceae were correlated with significant, visible fouling and pitting corrosion. This study identified the relationship between recurrent fouling of B20 with increased rates of corrosion, largely at the bottom of the sampled storage tanks.

microbiology↗

bioRxiv2026

Synbiotic Yogurt with Lactobacillus plantarum and Stevia rebaudiana: Physicochemical, Microbiological, and Functional Evaluation

Functional dairy products are increasingly recognized for their ability to provide both essential nutrition and additional health benefits. This study aimed to develop and evaluate a synbiotic yogurt enriched with Lactobacillus plantarum as a probiotic and Stevia rebaudiana extract (1% w/v) as a prebiotic source. Thirteen lactic acid bacteria (LAB) strains were isolated from fermented dairy and vegetable samples and evaluated for probiotic potential through tests for acid and bile tolerance, hydrophobicity, aggregation abilities, and pathogen co-aggregation. Isolate PG1 (Lactobacillus plantarum) demonstrated the highest prebiotic growth stimulation index (49%) in the presence of stevia extract and was selected for yogurt formulation. Yogurt samples were prepared and stored at 4{degrees}C for 10 days. Physicochemical properties (pH, titratable acidity, and protein content), microbiological viability, total phenolic and flavonoid content, antioxidant activity (DPPH assay), and sensory attributes were monitored. The synbiotic yogurt (St-Y) showed enhanced functional properties, with a total phenolic content of 16.67 {micro}g GAE/g, a flavonoid content of 6.28 {micro}g QE/g, and 57.84% antioxidant activity. Additionally, it showed improved protein content and superior sensory scores compared to control samples. These findings suggest that S. rebaudiana fortified probiotic yogurt can serve as a nutritious, antioxidant-rich, and sensory-acceptable functional dairy product.

microbiology↗

bioRxiv2026

Assessment of Microbial Consortia for Biogenic Mineralization Potential of Lead (Pb) using Microbiologically Induced Calcite Precipitation and Nitrate Reduction

Groundwater contamination by lead and nitrate exceeding the permissible limits of the World Health Organization (WHO) and the Bureau of Indian Standards (BIS) of 10 ppb lead and 45 to 50 ppm nitrate poses a severe health risk of systemic toxicity in humans and animals. Microbial nitrate reduction coupled with microbiologically induced calcite precipitation (MICP) proved to be a novel bioremediation strategy to transform soluble lead nitrate into insoluble lead biominerals. This study presents statistical optimization of operational parameters for nitrate reduction and co-precipitation of biogenic lead carbonate-calcium carbonate by nitrate-reducing bacterial consortia. The SEM, TEM with SEAD, and XRD analysis of the biomineral confirmed lead carbonate co-precipitation with CaCO3. A two-level factorial design was applied to screen significant variables and interactive factors that influence nitrate reduction and biomineral precipitation. The highest nitrate reduction, maximum ammonia production, and biogenic Pb-mineralization efficiency of > 90% were obtained at the optimum conditions (initial nitrate requirement of 600 ppm, temperature 37 oC, pH 8, and carbon content of 5.5 g/L) compared to those obtained under the common process parameters. ANOVA with a high correlation coefficient (R2 > 0.99) corroborated the linear model for the biogenic mineralization process. This study on the biogenic removal of Pb by nitrate-reducing bacterial consortia revealed possible opportunities for large-scale treatment of Pb-contaminated surface and subsurface water bodies.

microbiology↗

bioRxiv2015

Multi -omics and metabolic modelling pipelines: challenges and tools for systems microbiology

Integrated -omics approaches are quickly spreading across microbiology research labs, leading to i) the possibility of detecting previously hidden features of microbial cells like multi-scale spatial organisation and ii) tracing molecular components across multiple cellular functional states. This promises to reduce the knowledge gap between genotype and phenotype and poses new challenges for computational microbiologists. We underline how the capability to unravel the complexity of microbial life will strongly depend on the integration of the huge and diverse amount of information that can be derived today from - omics experiments. In this work, we present opportunities and challenges of multi -omics data integration in current systems biology pipelines. We here discuss which layers of biological information are important for biotechnological and clinical purposes, with a special focus on bacterial metabolism and modelling procedures. A general review of the most recent computational tools for performing large-scale datasets integration is also presented, together with a possible framework to guide the design of systems biology experiments by microbiologists.

Systems Biology↗

bioRxiv2020

Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index: a Rapid and Accessible Tool that Exploits Genomic Data in Public Health and Clinical Microbiology Applications

As microbial genomics makes increasingly important contributions to clinical and public health microbiology, the interpretation of whole genome sequence data by non-specialists becomes essential. In the absence of capsule-based vaccines, two protein-based vaccines have been used for the prevention of invasive serogroup B meningococcal disease (IMD), since their licensure in 2013/14. These vaccines have different components and different coverage of meningococcal variants. Hence, decisions regarding which vaccine to use in managing serogroup B IMD outbreaks require information about the index case isolate including: (i) the presence of particular vaccine antigen variants; (ii) the expression of vaccine antigens; and (iii) the likely susceptibility of its antigen variants to antibody-dependent bactericidal killing. To obtain this information requires a multitude of laboratory assays, impractical in real-time clinical settings, where the information is most urgently needed. To facilitate assessment for public health and clinical purposes, we synthesised genomic and experimental data from published sources to develop and implement the Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index, which is publicly-available on PubMLST (https://pubmlst.org). Using whole genome sequences or individual gene sequences obtained from IMD isolates or clinical specimens, MenDeVAR provides rapid evidence-based information on the presence and possible immunological cross-reactivity of different meningococcal vaccine antigen variants. The MenDeVAR Index enables practitioners who are not genomics specialists to assess the likely reactivity of vaccines for individual cases, outbreak management, or the assessment of public health vaccine programmes. MenDeVAR has been developed in consultation with, but independently of, both vaccine manufacturers.

genomics↗

bioRxiv2023

JAMS - A framework for the taxonomic and functional exploration of microbiological genomic data

Shotgun microbiome sequencing analysis presents several challenges to accurately and consistently depict sample composition and functional potential. Here we present a two-part framework - JAMS (Just a Microbiology System) - whereby with raw fastq files and metadata as input, meaningful analysis within a sample and between a sample can be performed with ease for either shotgun or 16S sequences. JAMS is the first package to provide seamless deconvolution of functions into their taxonomic contributors. We validated our JAMS framework on two human gut shotgun metagenome test datasets against the popular tool MetaPhlAn 4. We further demonstrate the application of the JAMS package, particularly the plotting functions, on a mouse shotgun dataset.

bioinformatics↗

bioRxiv2024

Clinical considerations on antimicrobial resistance potential of complex microbiological samples

Antimicrobial resistance (AMR) is one of our greatest public health challenges. Targeted use of antibiotics (AB) can reduce the occurrence and spread of AMR and boost the effectiveness of treatment. This requires knowledge of the antibiotic susceptibility (AS) of the pathogens involved in the disease. Therapeutic recommendations based on classical antibiotic susceptibility testing (AST) are based on the analysis of only a fraction of the bacteria present in the disease process. Next and third generation sequencing technologies allow the identification of antimicrobial resistance genes (ARGs) present in a bacterial community. Using this genomic approach, we can map the antimicrobial resistance potential (AMRP) of a complex, multi-bacterial microbial sample. The same approach can be used to identify antibiotics without any ARGs in the sample that interfere with their activity. Our paper summarises the clinical interpretation opportunities of genomic analysis results from 574 Escherichia coli strains and a complex microbiological sample from canine external otitis. In clinical metagenomics, AMRP may be an important approach to make AB therapy more targeted and effective. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/594174v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@d84733org.highwire.dtl.DTLVardef@12aa447org.highwire.dtl.DTLVardef@873811org.highwire.dtl.DTLVardef@1721aba_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2018

A Cross-Platform Comparison of Student’s Perceptions of the Learning Environment in an Introductory Microbiology Course

AbstractStudents positive perceptions of the learning environment increase retention and persistence in STEM disciplines. This study presents results from a Learning Environment Questionnaire administered at the beginning and end of the semester in a redesigned general/introductory microbiology course offered in traditional lecture, flipped and online sections. Split-sample t-tests and chi-squared tests were used for cross-section and within section analyses, respectively. The findings support the study hypothesis that student perceptions of the learning environment will vary as a function of platform. This work demonstrates the additional effect of the time in the semester when students complete the questionnaire and this effect on students perceptions over the course of the semester relative to their initial perceptions. The results of this study offer insight on student perceptions of the learning environment as universities embrace online education in introductory and gateway courses as a response to rising student enrollments and diminishing resources.

scientific communication and education↗

bioRxiv2019

Rock the Chalk: A five-year comparative analysis of a large microbiology lecture course reveals improved outcomes of chalk-talk compared to PowerPoint

The rise of electronic assisted presentation programs such as PowerPoint in undergraduate large lecture biology classes has displaced more traditional hand-drawn lectures such as the blackboard or overhead projectors, referred here as \"chalk-talk\" approaches. But which method is more effective in a large lecture microbiology classroom is unclear. Here I present data from a large microbial genetics lecture course taken during a five-year span comparing PowerPoint to chalk-talk lecturing methods. The results indicate that the chalk-talk approach was preferred by the students and rated higher in all measured metrics including course enjoyment, learning of key concepts, and course outcomes.

scientific communication and education↗

bioRxiv2016

Alternative Growth Behavior of Mycobacterium Avium Subspecies and Staphylococci with Implications for Clinical Microbiology and Blood Culture

Rapid culture of Mycobacterium avium subspecies paratuberculosis (MAP) from patients remains a challenge. During the process of developing a rapid culture method for MAP, we found that there is an alternative growth behavior present in MAP, MAH (Mycobacterium avium subspecies hominissuis) and other bacteria such as Staphylococcus aureus, and Staphylococcus pseudintermedius. The bacterial DNA, RNA and proteins are present in the supernatants of the liquid culture media after routine microcentrifugation. When cultured in the solid media plate, there are a limited number of colonies developed for MAP and MAH disproportionate to the growth. We believe there is an alternative growth behavior for MAP, MAH and other bacteria similar to \"phenoptosis\". Based on the alternative bacterial growth behavior, we tested 62 blood culture specimens that have been reported negative by routine automated blood culture method after 5 days of incubation. We used alternative culture media and molecular diagnostic techniques to test these negative culture bottles, and we found a large percentage of bacterial growth by alternative culture media (32%) and by molecular PCR amplification using 16s rDNA primer set and DNA sequencing (69%). The sensitivity of detection by the molecular PCR/sequencing method is significantly higher than by routine automated blood culture. Given the challenge of early diagnosis of sepsis in the hospital setting, it is necessary to develop more sensitive and faster diagnostic tools to guide clinical practice and improve the outcome of sepsis management.

Microbiology↗

bioRxiv2020

Rapidly predicting vancomycin resistance of Enterococcus faecium through MALDI-TOF MS spectra obtained in real-world clinical microbiology laboratory

Enterococcus faecium is one of the leading pathogens in the world. In this study, we proposed a strategy to rapidly and accurately distinguish vancomycin-resistant Enterococcus faecium (VREfm) and vancomycin-susceptible E. faecium (VSEfm) to help doctors correctly determine the use of vancomycin by a machine learning (ML)-based algorithm. A predictive model was developed and validated to distinguish VREfm and VSEfm by analyzing MALDI-TOF MS spectra of unique E. faecium isolates from different specimen types. Firstly, 5717 mass spectra, including 2795 VREfm and 2922 VSEfm, were used to develop the algorithm. And 2280 mass spectra of isolates, namely 1222 VREfm and 1058 VSEfm, were used to externally validate the algorithm. The random forest-based algorithm demonstrated good classification performances for overall specimens, whose mean AUROC in 5-fold cross validation, time-wise validation, and external validation was all greater than 0.84. For the detection of VREfm in blood, sterile body fluid, urinary tract, and wound, the AUROC in external validation was also greater than 0.84. The predictions with algorithms were significantly more accurate than empirical antibiotic use. The accuracy of antibiotics administration could be improved by 30%. And the algorithm could provide rapid antibiotic susceptibility results at least 24 hours ahead of routine laboratory tests. The turn-around-time of antibiotic susceptibility could be reduced by 50%. In conclusion, a ML algorithm using MALDI-TOF MS spectra obtained in routine workflow accurately differentiated VREfm from VSEfm, especially in blood and sterile body fluid, which can be applied to facilitate the clinical testing process due to its accuracy, generalizability, and rapidness.

microbiology↗

bioRxiv2020

Microbiological safety of industrially reared insects for food: Identification of bacterial endospores and targeted detection of foodborne viruses

Edible insects are characterised by high microbial numbers of which the bacterial endospores fraction can survive (thermal) processing. It is unknown, however, which bacterial species occur as endospore in edible insects and what impact they have on food safety. Additionally, edible insects have never been explored for the presence of foodborne viruses so far. In this study, we found that the bacterial endospore fraction in a collection of mealworm and cricket samples obtained from commercial insect producers can comprise a large amount of Bacillus cereus group members that can pose insect or human health risks. Monitoring and effective mitigation of these risks are urged. By contrast, none of the foodborne viruses hepatitis A virus, hepatitis E virus and norovirus genogroup II were detected in the sample collection. Therefore, food safety risks originating from these viral pathogens can be concluded to be low.

microbiology↗