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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.

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.

We present a new approach to segment and classify bacterial spore layers from Transmission Electron Microscopy (TEM) images using a hybrid Convolutional Neural Network (CNN) and Random Forest (RF) classifier algorithm. This approach utilizes deep learning, with the CNN extracting features from images, and the RF classifier using those features for classification. The proposed model achieved 73% accuracy, 64% precision, 46% sensitivity, and 47% F1-score with test data. Compared to other classifiers such as AdaBoost, XGBoost, and SVM, our proposed model demonstrates greater robustness and higher generalization ability for non-linear segmentation. Our model is also able to identify spores with a damaged core as verified using TEMs of chemically exposed spores. Therefore, the proposed method will be valuable for identifying and characterizing spore features in TEM images, reducing labor-intensive work as well as human bias.

Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.