Journal of Microbiological Methods

The minimum inhibitory concentration (MIC) is a standard measure for describing the lowest effective dose concentration of an antimicrobial compound in clinical practice; yet, conventional assays often require a substantial amount of antimicrobial compound, limiting their use with scarce, purified agents. Here, we describe a simple and reproducible technique to evaluate the MIC for purified compounds with a limited sample size. The protocol describes the MIC steps against a bacterial strain while minimizing the use of reagents and materials. It is helpful for screening purified natural products as antimicrobial agents and in early-stage drug discovery. The protocol adapts standard microplate-based assays for two-fold dilution of the compound, ensuring their applicability in microbiological studies. The MIC value of the standard antibiotic kanamycin against Staphylococcus aureus, Vibrio fischeri, Klebsiella pneumoniae, and Escherichia coli was determined using our method, and was found to be consistent with the conventional broth microdilution method, validating its reliability. Therefore, this method offers a practical and viable solution for antimicrobial drug discovery, addressing the disparity between limited compound availability and comprehensive microbiological assessment of MIC.

With the rapid expansion of global food demand, aquaculture has become a critical pillar for future food security. However, aquaculture systems remain highly vulnerable to pathogenic bacteria, and rapid identification of antagonistic microbes is essential for sustainable disease control. Conventional evaluation approaches rely on fluorescence labeling or post-culture assays, limiting the ability to quantify dynamic interactions in mixed microbial populations in a real-time and label-free manner. Here, we propose a computational framework for classifying the mixing ratio of Vibrio harveyi and environmental bacteria using time-series motion features extracted from microscopy videos. We defined 24 interpretable motility descriptors and employed a Temporal Convolutional Network (TCN) to learn their temporal structure. The proposed method achieved a classification accuracy of 93.3%, outperforming conventional static statistical approaches and alternative machine learning models. These findings indicate that mixture discrimination in microbial communities is governed not by absolute motility magnitude, but by collective alignment and its temporal stability. Our study establishes a time-resolved computational framework for quantifying dynamic collective order in mixed microbial populations and highlights its potential for label-free automated screening and robotic microbiological applications.

The coprophilous agaricomycete Coprinopsis cinerea is a model organism for antagonistic fungal-bacterial interactions. Previous studies showed that C. cinerea responds to antagonistic bacteria with strong induction of a set of genes encoding secreted antibacterial molecules. However, little is known about the elicitors of this response. Key open questions in this respect include whether individual antibacterial defence genes are induced by different bacteria and/or by specific bacterial soluble molecules. Here, we present a new C. cinerea reporter system to monitor antibacterial defence induction and address related outstanding issues with minimal hands-on time. In this system, the promoter of the endogenous bacterial-induced gene cclys1 drives the expression of cnluc, which encodes a secreted variant of the deep-sea shrimp luciferase Nluc. We show that cNluc allows to detect and quantify cclys1 induction by measuring luminescence directly in the culture medium of reporter strain colonies. Building on these features, we successfully leveraged the inducible cNluc reporter strain for the development of a novel 96-well plate assay that allows the high-throughput screening of antibacterial defence elicitors. As cNluc can be subject to degradation by secreted proteases of fungal or bacterial origin in the culture medium, we coupled this assay to confirmatory qRT-PCR. Testing this set-up by confronting the reporter strain with several different bacteria revealed that cclys1 induction occurs independently of the bacterial ecological niche. Based on these results, we also recommend qRT-PCR exclusively for validation of negative results. We conclude that cNluc offers significant advantages over cytoplasmic reporter proteins, especially for preliminary rapid screening of multiple conditions.

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.

Mimiviral polymerase X, mvPolX, is a repair polymerase that is involved in base excision repair (BER) and carries out the gap-filling function in double-stranded DNA (dsDNA). We demonstrate a sensitive and sequence-specific DNA detection method using this polymerase. mvPolX begins polymerizing DNA from the 3 end of a gap, displacing the downstream nucleotides without exonuclease activity. Our detection method is built on this activity of mvPolX. We designed a probe molecule consisting of a partial dsDNA with a 3 over-hang region complementary to the target DNA to be detected. The probe has a fluorophore-quencher (FAM-BHQ1) tag to facilitate detection upon strand removal. Binding of the probe to the complementary target forms a dsDNA with a single nucleotide gap in one strand. mvPolX binds this gap region and begins polymerisation eventually displacing the quencher strand leading to an increase in fluorescence. Proof-of-concept has been established using a synthetic 19 bp target DNA sequence. The method is specific and did not show any strand displacement when a single or double mismatched nucleotide at the 3 end of the target DNA was used. To demonstrate this molecular assay, we used M13 phage as our target. Asymmetric PCR (aPCR) was used to obtain single-stranded target DNA (158 bases) from M13 genomic DNA, which was directly used in the assay as target. The combination of aPCR and mvPolX assay can detect as low as 10 copies of genomic DNA. The enzymatic reaction is fast, requiring only 15 min of incubation with mvPolX at 30 {degrees}C. We have further demonstrated the efficiency of the assay in presence of multiple non-target DNA by detecting the target DNA from M13 phage spiked lakewater samples.

Probiotics produce antimicrobial peptides and small molecules that are secreted into the medium. Antimicrobial activity of cell-free supernatants can be tested using various qualitative and quantitative methods. Many of these techniques employ methods which introduce uncontrolled variables, impacting reproducibility and making comparison of reported results difficult. Here, we present a simple procedure for quantitative estimation of antimicrobial activity of cell-free supernatants which overcomes drawbacks of commonly used methods.

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

Melanins are biological macromolecule with immense functionality synthesised by a wide spectrum of living organism. It is mainly synthesised by the oxidative polymerization of indolic and phenolic compounds through several enzymatic process. It has wide spread application in agriculture, cosmetic and therapeutic industry due to its various properties including antioxidation ability, UV protection efficiency and anticancer activity. Because of this wide range of application in different sectors, large scale production and commercialization attains enormous consideration. The present study deals with the effect of 12 different process parameters on melanin production viz., production media, incubation time, inoculum concentration, pH, temperature, agitation, carbon source, phosphate and magnesium source, CuSO4.5H2O, sodium chloride and L-tyrosine on melanin production by Pseudomonas stutzeri strain BTCZ 109 obtained from Arabian sea sediments was evaluated. After optimizing the important process parameters, the bacteria showed about ~4.65 fold increase in melanin production compared to unoptimized cultural conditions. The melanin optimized through this method was found to be nano sized. The Nano sized DOPA melanin in treating Skin cancer cell line SK ML28 which showed a dose-dependent activity with an IC50 value of 164 g/mL. All these results highlight the therapeutic efficiency of DOPA melanin Nano particle as promising bioactive molecule.

Culture conditions were optimized for the production of melanin nanoparticle by the bacterial strain Pseudomonas stutzeri BTCZ 305. Response surface methodology was employed for determining the most significant fermentation conditions using variables including, pH, temperature and L-tyrosine concentration identified through one-factor-at-a time approach. Box-behnken design consisting of 17 different combinations of all these factors were performed. Using this methodology, a quadratic regression model was built and the optimal combinations of media constituents for maximum melanin production 1192.27 microg/mL were determined as temperature (32.5 degreeC), pH (8.5) and L-tyrosine concentration (7 g/L). Melanin production was obtained experimentally coincident with the predicted value and the model was proven to be adequate. The nanostructural distribution, its stability in colloidal suspension and particle size were also characterized with the help of TEM, particle size analysis and Zeta potential. The potent applicability of this molecule in anti-inflammation and wound healing was also elucidated.

To explore adapting qPCR systems for end-point nucleic acid quantification using dyes such as SYTO-9, we quantified serial dilutions of DNA and RNA standards in the range of 0.75 - 200 ng/{micro}l on 384-well qPCR devices. SYTO-9 fluorescence was successfully measured using standard SYBR Green settings. Blank-subtracted relative SYTO-9 signal showed a logarithmic dependence on DNA/RNA concentration (R2 > 0.95). Measurements were highly stable with different incubation times, temperatures of up to 95{degrees}C, and photobleaching. The described approach is a valuable QC option for high-throughput DNA/RNA isolations and could be adapted to additional fluorometric assays beyond nucleic acids.

The effect of microcurrents on facultative microbial pathogens remains controversial. Solid cultures in Sabouraud Glucose Agar of the ubiquitous mold Aspergillus fumigatus were repeatedly treated with a commercially available device performing wireless conductive microelectrostimulation by 3.5 A microcurrent routed by spraying negatively charged air particles onto solid cultures in modified petri dishes. The treated cultures displayed increased growth compared to standard ones, but only as a function of mycelial density and total surface; the radial growth rate of the mycelium remained unaltered. The increased growth was positively related to the duration of the treatment. At the same time, secondary development (new mycelial loci within the dish) was greatly upheld due to treatment, as the spraying created microairstreams dislocating the fungal spores. These results imply perplexed kinetics of mycelial growth both with and without treatment, since the folding of the mycelial mat is observed regularly. Both the fungus response to the ES and the possible revision of growth kinetics create prospects for biotechnological and bioremediation applications but also imply biomedical considerations, regarding infection dynamics of mycelial fungi and their in situ resistance to immune responses and treatment.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease gene (Cas), originating from the bacteria acquired immune system, have revolutionized gene editing technology. In this regard, type II (Cas9) been extensively studied and widely applied CRISPR system so far. The mechanism for precise manipulation of genomic sequences is guided by small RNA called CRISPR RNA (crRNA). In this study we devised and optimized CRISPR-Cas9 screening system based on Cas9 gene detection, targeting a conserved part of recognition domain (REC) consisting of arginine rich bridge helix (BH). We used hemi-nested PCR approach for screening sensitivity and reproducibility. The recombinant E. coli DH5 alpha containing the pRGEB32 vector (DH5 alpha/pRGEB32) with the Cas9 gene was used for system optimization. Subsequently, the screening system was applied and validated on different environmental bacterial strains including Alcaligenes faecalis and Pseudomonas stutzeri, isolated from sewerage samples. The optimized hemi-nested PCR resulted in amplification of targeted region in environmental bacterial strains and results were reproduced successfully. Furthermore, nucleotides and amino acid sequence, motif and domain analysis of PCR products, confirmed the targeted Cas9 REC-BH domain. Presently, no rapid and cost effective CRISPR-Cas screening system is available except expensive whole genome sequencing approach. Our investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species. In this context, the developed Cas9 gene-based CRISPR-Cas screening system (C9CSS) may be a potential rapid screening tool to identify new Cas9 orthologs in different bacterial genomes with improved functions.

Bacterial cellulose (BC) is a biopolymer that comes from natural sources. It has high purity, crystallinity, mechanical strength, and biocompatibility, which makes it suitable for various biomedical and industrial uses. Unlike plant cellulose, BC does not contain lignin or hemicellulose. This results in better material quality and simpler processing. However, producing BC on a large scale is limited by high production costs, expensive culture media, and dependence on a few bacterial strains. It is essential to find cost-effective production methods and alternative microbial sources to expand its commercial use. This study looked at the cellulose-producing ability of Acetobacter diazotrophicus, a safe and relatively unexplored bacterium, under different growth conditions. We compared bacterial growth and cellulose production using Hestrin-Schramm (HS) medium, the standard for BC production, and LB supplemented with glucose (LB+Glucose), which we explored as a more affordable option. We analyzed growth rates, inoculum age, and pH levels to find the best conditions for cellulose production. We observed faster bacterial growth in HS medium, with a doubling time of 3.906 hours, compared to 6.241 hours in LB+Glucose medium. Cellulose production was greatly affected by inoculum age, with successful synthesis from cultures that were agitated for 36 to 40 hours. The highest cellulose yield was at pH 6.0 in HS medium (4.6 mg/mL) and at pH 5.5 in LB+Glucose medium (3.6 mg/mL). FTIR analysis confirmed the presence of characteristic functional groups of bacterial cellulose. These results suggest that Acetobacter diazotrophicus is a promising and cost-effective option for producing bacterial cellulose and highlight the importance of medium composition, inoculum age, and pH for optimizing production.

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d92fe5org.highwire.dtl.DTLVardef@d76c07org.highwire.dtl.DTLVardef@f5516forg.highwire.dtl.DTLVardef@1b5864b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

Health monitoring is essential to ensure laboratory animal welfare and the reliability of experimental data in zebrafish research facilities. Conventional surveillance strategies based on resident or sentinel fish have limitations in terms of sensitivity and animal use, highlighting the need for alternative approaches consistent with the 3Rs principles. In this perspective, the present study evaluated the use of sludge collected from recirculating aquaculture systems as an environmental matrix for molecular health monitoring. Because sludge accumulates microorganisms and organic material from the entire system, it represents a promising sample for pathogen detection. Following an initial environmental surveillance phase to detect pathogens present in the system, the study aimed to optimise a molecular protocol for sludge analysis. To this end, four commercial DNA extraction kits were evaluated to assess their effectiveness in recovering bacterial DNA from sludge. Their performance was analysed by quantitative PCR in terms of extraction efficiency, repeatability, and limit of detection. The results highlighted the strengths and limitations of each DNA extraction protocol and confirmed the suitability of sludge as a non-lethal matrix for pathogen detection. These findings support the implementation of environmental monitoring as a practical and cost-effective alternative to sentinel-based surveillance, improving pathogen detection while reducing animal use in accordance with the 3Rs principles and Directive 2010/63/EU.

Petri dishes are routinely photographed using handheld cameras under reflected light. While adequate for large, distinct colonies, this approach fails to capture individual organisms or colonies that are transparent, do not reflect light well, or have fine structures, and is unsuitable for high quality timelapses. Darkfield illumination provides superior contrast for organisms on agar surfaces, but existing solutions rely on improvised setups with inconsistent lighting, or on large and expensive benchtop instruments that cannot be housed inside incubators. We present DishCam, an open-source device consisting entirely of 3D-printed parts and inexpensive off-the-shelf electronics, controlled by a Raspberry Pi running custom open-source software. DishCam produces high-contrast darkfield images using angled LED illumination beneath a Petri dish. All components used are off-the-shelf or easily 3D-printed. The camera and lights can be fully controlled through Raspberry Pi using our software. We demonstrate the device across multiple biological systems: Klebsiella aerogenes colonies of differing densities, Trypanosoma brucei colonies doing social motility, Dictyostelium discoideum streaming aggregation, and Caenorhabditis elegans on feeding substrates. The total cost of the basic device is approximately {pound}166 or $222, with the upgraded configuration costing approximately {pound}413 or $552, which includes a high-quality telephoto lens, SSD storage and incubator-compatible cables.

BackgroundInnovative vector control strategies are urgently needed to combat malaria transmission by Anopheles gambiae, given the limitations of current insecticides--namely resistance, human toxicity, environmental damage, and high costs. This study evaluated the larvicidal efficacy of endophytic bacteria isolated from Cola nitida against third-instar larvae of Anopheles gambiae. MethodsEndophytic bacteria were isolated from nine Cola nitida plant parts (leaves, roots, stems, fruits, flowers, stem bark, branches, root bark, whole plant extracts) using standard surface sterilization and culturing protocols. Thirty-two morphologically distinct strains were purified and screened for larvicidal activity against third-instar An. gambiae larvae using the WHO-recommended turbidity method at McFarland 4 standard. Six active isolates underwent dose-response testing (McFarland 0.5-4) with mortality recorded at 24 and 48 hours. Environmental safety was assessed via Lemna minor growth inhibition, and molecular identification employed the API 20E biochemical gallery. ResultsScreening identified six strains with significant larvicidal activity (LC < McFarland 2). At McFarland 2 concentration, all six strains achieved >50% mortality within 24-48 hours, with Lemna minor EC >100% indicating minimal phytotoxicity. API 20E identification (96-99.99% similarity) revealed three strains (6164, 6211, 6501) matching Photobacterium damselae, two (6186-1, 6512-1) matching Salmonella enterica subsp. arizonae, and one (6600) matching Pseudomonas sp., confirmed by distinctive biochemical profiles. ConclusionThis study provides the first evidence of potent, environmentally safe larvicidal activity from Cola nitida endophytic bacteria against Anopheles gambiae. These strains represent promising biocontrol candidates to address insecticide resistance, offering a sustainable alternative for malaria vector management in endemic regions.

Cronobacter sakazakii is an opportunistic foodborne pathogen that affects neonates, and has ability to produce dry surface biofilm (DSB). However, little is known about its resistance to dry sanitizers. This study aimed to evaluate the efficiency of dry sanitizers compared with sodium hypochlorite (SH) on DSB of C. sakazakii. DSB were formed on stainless steel or polypropylene coupons (10 cm{superscript 2}) using two cycles of hydrated/dry phases at 25 {degrees}C: T1 (48/48 h) or T2 (24/120 h). At DSB endpoint, the coupons were sanitized with 70% ethanol, a commercial product based on isopropyl alcohol (25%) and quaternary ammonium (0.015%), hot air (90{+/-}2 {degrees}C), UV-C light (254 nm), gaseous ozone (45{+/-}2 mg/L), or SH (200 mg/L, pH 6.5). All dry sanitizers showed limited antimicrobial activity (p > 0.05), with reductions [&le;] 0.8 log CFU/cm{superscript 2} over 30 min exposure. In contrast, SH was effective against DSB regardless of the DSB formation protocol, with reductions [&ge;]2 and >5 log CFU/cm{superscript 2} after 10 and 30 min, respectively. Interestingly, DSB formed with shorter hydrated phase and longer dry phase (T2) had greater sensitivity to SH, but it was not noted to dry sanitizers. CLSM images suggested the presence of VBNC cells, particularly after SH treatment. In conclusion, our results emphasize the importance of implementing stringent hygiene measures to control C. sakazakii DSB in the low moisture food industry, and indicate that SH is an effective sanitation strategy when the drying line is promptly addressed.

Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus, and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs. Author SummaryStaphylococcus aureus is a bacterial species naturally present in our external flora and environment, but it is also one of the main pathogens responsible for nosocomial infections in hospital, with strains having highly problematic multi-resistance to antibiotics. S. aureus is able to secrete various virulence factors, some of which can specifically target and lyse our immune cells, making us more vulnerable to this pathogen. Thus, leukotoxins bind to receptors on the cell surface, drastically change their conformation and form cytotoxic pores in the membrane. Studying the molecular mechanisms underlying the formation of these pores is technically challenging due to their requirement for specific receptors. Here, we tested a simple electrophysiological method enabling the real-time measurement of pore formation on model cells (Xenopus oocytes), which express the receptors of interest. We were thus able to elucidate the kinetics of pore formation, the limiting role of receptors in this process, and propose a complementary model to the standard model. We also demonstrated the ability of this method to detect pore formation of non-cognate pairs of subunits and suggest further applications to characterize pore-forming properties of other toxins, to identify new target receptors, or to screen therapeutic agents inhibiting the formation of pores.

Quantitative microscopy of microorganisms increasingly produces large, multidimensional datasets, yet their analysis often depends on fragmented workflows spanning file conversion, segmentation, quality control, fluorescence quantification, tracking, and visualization. Here, we present BactoMate, an open-source, cross-platform graphical user interface that integrates these steps into a unified workflow for microbial image analysis. BactoMate incorporates established segmentation methods and supports both single-file and batch processing. Its modules enable image preprocessing, cell segmentation, morphology-based quality control, fluorescence and foci quantification, single-cell tracking, lineage reconstruction, structured data export, and generation of quality-control and visualization outputs. We demonstrate the applicability of BactoMate across multichannel fluorescence imaging, bacterial swimming assays, microcolony lineage analysis, phage infection assay and a microfluidic time series. All user-configurable parameters are exposed through the interface, are recorded alongside structured outputs and can be loaded for reproducible image analyses across experiments to reduce introduction of bias. By reducing workflow handoffs while preserving parameter control and exportable results, BactoMate enables accessible, reproducible, and scalable quantitative analysis of microbial microscopy data.