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Growing biofilms of thermophilic (heat-loving) and psychrotrophic (cold-tolerant) bacteria pose several challenges due to specific environmental requirements. Thermophilic bacteria typically grow between 45 and 80 C, while psychrotrophic bacteria thrive between 0 and 15 C. Maintaining the precise temperature and fluid conditions required for biofilm growth can be technically challenging. To overcome these challenges, we designed the Bio-Rocker, a temperature- and shear stress-controlled rocker platform for biofilm incubation. The platform supports temperatures between − 9 and 99 C, while its digital controller can adjust the rocking speed from 1 to 99/s and set rocking angles up to ±19. This ability, together with data from analytical models and multi-physics simulations, provides control over the shear stress distribution at the growth surfaces, peaking at 2.4 N/m. Finally, we evaluated the system’s ability to grow bacteria at different temperatures, shear stress, and materials by looking at the coverage and thickness of the biofilm, as well as the total biomass. A step-by-step guide, 3D CAD files, and controller software is provided for easy replication of the Bio-Rocker, using mostly 3D-printed and off-the-shelf components. We conclude that the Bio-Rocker’s performance is comparable to high-end commercial systems like the Enviro-Genie (Scientific Industries) yet costs less than $350 dollars to produce.

Analyzing microscopy images of large growing cell samples using traditional methods is a complex and time-consuming process. In this work, we have developed an attention-driven UNet-enhanced model using deep learning techniques to efficiently quantify the position, area, and circularity of bacterial spores and vegetative cells from images containing more than 10,000 bacterial cells. Our attention-driven UNet algorithm has an accuracy of 96%, precision of 82%, sensitivity of 81%, and specificity of 98%. Therefore, it can segment cells at a level comparable to manual annotation. We demonstrate the efficacy of this model by applying it to a live-dead decontamination assay. The model is provided in three formats: Python code, a Binder that operates within a web browser without needing installation, and a Flask Web application for local use.

Purpose: Hypochlorite-based formulations are widely used for surface disinfection. However, the efficacy of hypochlorite against spore-forming bacteria varies significantly in the literature. Although neutral or low pH hypochlorite solutions are effective sporicides due to the formation of hypochlorous acid (HOCl), their optimal conditions and the specific role of pH in disinfection remain unclear. These conditions also increase the solution’s corrosiveness and compromise its shelf life. Therefore, further research is needed to identify the pH conditions that balance solution stability and effective hypochlorite-based spore disinfection.

Results: This study investigates the impact of neutral to alkaline pH on the sporicidal efficiency of hypochlorite against a pathogenic Bacillus cereus strain. We apply a 5,000 ppm hypochlorite formulation for 10-min across a pH range of 7.0-12.0, simulating common surface decontamination practices. Our results demonstrate that hypochlorite is largely ineffective at pH levels above 11.0, showing less than 1-log reduction in spore viability. However, there is a significant increase in sporicidal efficiency between pH 11.0 and 9.5, with a 4-log reduction in viability. This pH level corresponds to 2 - 55 ppm of the HOCl ionic form of hypochlorite. Further reduction in pH slightly improves the disinfection efficacy. However, the shelf life of hypochlorite solution decreases exponentially below pH 8.5. To explore the pH-dependent efficacy of hypochlorite, Raman spectroscopy and fluorescence imaging were used to investigate the biochemical mechanisms of spore decontamination. Results showed that lower pH enhances spore permeability and promotes calcium dipicolinic acid (CaDPA) release from the core.

Conclusion: Our results highlight the complex relationship between pH, sporicidal efficacy of hypochlorite, and its shelf life. While lower pH enhances the sporicidal efficiency, it compromises the solution’s shelf life. A pH of 9.5 offers a balance, significantly improving shelf life compared to previously suggested pH ranges 7.0-8.0 while maintaining effective spore inactivation. Our findings challenge the common practice of diluting sodium hypochlorite with water to a 5,000 ppm solution, as this highly alkaline solution (pH of 11.9), is insufficient for eliminating B. cereus spores, even after a 10-min exposure. These findings are critical for improving disinfection practices, highlighting the importance of optimizing sodium hypochlorite effectiveness through pH adjustments before application.

Spores of species belonging to the Bacillus cereus sensu lato (s.l.) group are common contaminants in food processing environments due to their ability to adhere to surfaces and resist cleaning procedures. These spores are equipped with pilus-like endospore appendages (ENAs), which are believed to promote surface adhesion. We investigated the role of ENAs in spore adhesion to abiotic surfaces using a wild-type (WT) Bacillus paranthracis strain and isogenic mutants lacking ENAs or an intact exosporium. WT spores expressing both short and long ENAs (S+L+) adhered significantly more to stainless steel (SS) and polypropylene (PP) compared to bald spores (S-L-) and spores of an exosporium-deficient mutant (Delta exsY), whereas adhesion to polystyrene (PS) and glass was not significantly affected by the presence of ENAs. The Delta exsY mutant also showed the lowest adhesion across all tested surfaces, a pattern similarly observed for vegetative cells. The strongest adhesion to PP was observed when both fiber types were present. A clear trend also emerged: on PP, WT remained adhered for at least an hour, while bald spores tended to detach within that time. Under saline conditions and at different pH levels, bald spores adhered strongly to SS. However, in the presence of a non-ionic surfactant or a concentrated protein solution, WT spores adhered more. Our results highlight the crucial role of ENAs in B. cereus spp. spore adhesion to industrially relevant surfaces, providing mechanistic insight into spore persistence. These insights support the design of surface treatments to prevent contamination, spoilage, and foodborne illnesses.IMPORTANCEBacteria belonging to the Bacillus cereus sensu lato group represent a persistent challenge in food production due to their highly resilient endospores (spores), which withstand cleaning, disinfection, and food processing. Understanding spore adhesion is essential for designing effective surface treatments that reduce chemical use, enhance food safety and quality, and minimize environmental impact. This study underscores the important role of endospore appendages (ENAs) in spore adhesion to common materials in food processing and laboratory environments. Wild-type spores expressing both S-ENA and L-ENA adhered significantly more than mutants lacking ENAs or the exosporium, highlighting ENAs as potential targets for disrupting spore adhesion. Time-dependent adhesion assays on polypropylene revealed strong, sustained attachment by wild-type spores, contrasting with weaker, transient adhesion by ENA-depleted mutants. These findings offer valuable insights into B. paranthracis spore adhesion dynamics, guiding the development of tailored cleaning protocols to improve contamination control and sustainability.

Raman spectroscopy is a highly informative technique that is routinely used to analyze the chemical bonds in molecules, such as peptides. However, while Raman spectra of aqueous single amino acids are well-characterized, proteins and peptides are usually found only in solutions in the presence of ions for stability or further analysis. The impact of ions on the Raman spectra of amino acids and subsequently on peptides and proteins has not been systematically studied. In this work, the impact of different metal ions on the Raman spectra of four significant amino acids (glutamate, tyrosine, cysteine, and serine) and N-methylacetamide (a model of a peptide bond) in aqueous solution was studied using density functional theory simulations and Raman spectroscopy experimental validation. The research focused on analyzing band shifts and variations in peak intensity when different cations commonly used in experiments (Li+, Na+, K+, Ag+, Au+, Cu+, and Cu2+) were present. Experimental evidence on Raman spectra variation of Glutamate in the presence of different concentrations of Cu2+is also provided. Our results show that the ions stay close to the oxygen atoms in the amino acids tested. With regard to Raman spectra, we show Cu2+promotes Raman resonance. We have characterized and tabulated the shifts in the characteristic Raman peaks of the amino acids in response to metal ions with the aim of providing a reference for interpreting Raman spectra of proteins and other biological materials.

Bacterial spores are highly resilient and capable of surviving extreme conditions, making them a persistent threat in contexts such as disease transmission, food safety, and bioterrorism. Their ability to withstand conventional sterilization methods necessitates rapid and accurate detection techniques to effectively mitigate the risks they present. In this study, we introduce a surface-enhanced Raman spectroscopy (SERS) approach for detecting Bacillus thuringiensis spores by targeting calcium dipicolinate acid (CaDPA), a biomarker uniquely associated with bacterial spores. Our method uses probe sonication to disrupt spores, releasing their CaDPA, which is then detected by SERS on drop-dried supernatant mixed with gold nanorods. This simple approach enables the selective detection of CaDPA, distinguishing it from other spore components and background noise. We demonstrate detection of biogenic CaDPA from concentrations as low as 10³ spores/mL, with sensitivity reaching beyond CaDPA levels of a single spore. Finally, we show the method’s robustness by detecting CaDPA from a realistic sample of fresh milk mixed with spores. These findings highlight the potential of SERS as a sensitive and specific technique for bacterial spore detection, with implications for fields requiring rapid and reliable spore identification.

Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm−1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm−1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.

Scanning electron microscopy (SEM) can reveal the ultrastructure of bacterial spores, including morphology, surface features, texture, spore damage, germination, and appendages. Understanding these features can provide a basis for adherence, how physical and environmental stressors affect spore viability, integrity, and functionality, as well as the distribution and function of surface appendages. However, the spore sample preparation method can significantly impact the SEM images' appearance, resolution, and overall quality. In this study, we compare different spore preparation methods to identify optimal approaches for preparation time, spore appearance and resolved features, including the exosporium and spore pili, for SEM imaging. We use Bacillus paranthracis as model species and evaluate the efficacy of preparation protocols using different fixation and drying methods, as well as imaging under room- and cryogenic temperatures. We compare and assess method complexity to the visibility of the spore exosporium and spore appendages across different methods. Additionally, we use Haralick texture features to quantify the differences in spore surface appearance and determine the most suitable method for preserving spore structures and surface features during SEM evaluation. The findings from this study will help establish protocols for preparing bacterial spores for SEM and facilitating accurate and reliable analysis of spores' characteristics.

Species within the Bacillus cereus sensu lato group, known for their spore-forming ability, are recognized for their significant role in food spoilage and food poisoning. The spores of B. cereus are adorned with numerous pilus-like appendages, referred to as S-ENAs and L-ENAs. These appendages are thought to play vital roles in self-aggregation, adhesion, and biofilm formation. Our study investigates the role of S-ENAs and L-ENAs, as well as the impact of various environmental factors on spore-to-spore contacts and the interaction between spores and vegetative cells, using both bulk and single-cell approaches. Our findings indicate that ENAs, especially their tip fibrillae, play a crucial role in spore self-aggregation, but not in the adhesion of spores to vegetative cells. The absence of L-BclA, which forms the L-ENA tip fibrillum, reduced spore aggregation mediated by both S-ENAs and L-ENAs, highlighting the interconnected roles of S-ENAs and L-ENAs. We also found that increased salt concentrations in the liquid environment significantly reduced spore aggregation, suggesting a charge dependency of spore-spore interactions. By shedding light on these complex interactions, our study offers valuable insights into spore dynamics. This knowledge can inform future studies on spore behaviour in environmental settings and assist in developing strategies to manage bacterial aggregation for beneficial purposes, such as controlling biofilms in food production equipment.

The ability to detect and inactivate spore-forming bacteria is of significance within, for example, industrial, healthcare, and defense sectors. Not only are stringent protocols necessary for the inactivation of spores but robust procedures are also required to detect viable spores after an inactivation assay to evaluate the procedure’s success. UV radiation is a standard procedure to inactivate spores. However, there is limited understanding regarding its impact on spores’ spectral and morphological characteristics. A further insight into these UV-induced changes can significantly improve the design of spore decontamination procedures and verification assays. This work investigates the spectral and morphological changes to Bacillus thuringiensis spores after UV exposure. Using absorbance and fluorescence spectroscopy, we observe an exponential decay in the spectral intensity of amino acids and protein structures, as well as a logistic increase in dimerized DPA with increased UV exposure on bulk spore suspensions. Additionally, using micro-Raman spectroscopy, we observe DPA release and protein degradation with increased UV exposure. More specifically, the protein backbone’s 1600–1700 cm^–1 amide I band decays slower than other amino acid-based structures. Last, using electron microscopy and light scattering measurements, we observe shriveling of the spore bodies with increased UV radiation, alongside the leaking of core content and disruption of proteinaceous coat and exosporium layers. Overall, this work utilized spectroscopy and electron microscopy techniques to gain new understanding of UV-induced spore inactivation relating to spore degradation and CaDPA release. The study also identified spectroscopic indicators that can be used to determine spore viability after inactivation. These findings have practical applications in the development of new spore decontamination and inactivation validation methods.