Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and intercellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cellular proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane-potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely Bacillus subtilis and Escherichia coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5-second electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K⁺ and the deletion of the K⁺ channel suggested that the hyperpolarization response is caused by the K⁺ efflux through the channel. When cells are preexposed to 400 ± 8 nm wavelength light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh–Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore, or alcohol. Therefore, electrically induced membrane-potential dynamics offer a reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level. Significance Transmembrane voltage in bacteria plays a central role both in electrical signaling and cell proliferation. However, whether proliferation state influences the response to electrical signals was unknown. Combining fluorescence time-lapse microscopy with a bespoke device and mathematical modeling, we show that proliferative and inhibited cells respond in opposite directions to an identical electrical signal. The response differentiation can be seen within a minute after stimulation. Therefore, our findings offer an approach for rapid detection of proliferative bacteria at the single-cell level.

Background: Clostridioides difficile is a spore forming bacterial species and the major causative agent of nosocomial gastrointestinal infections. C. difficile spores are highly resilient to disinfection methods and to prevent infection, common cleaning protocols use sodium hypochlorite solutions to decontaminate hospital surfaces and equipment. However, there is a balance between minimising the use of harmful chemicals to the environment and patients as well as the need to eliminate spores, which can have varying resistance properties between strains. In this work, we employ TEM imaging and Raman spectroscopy to analyse changes in spore physiology in response to sodium hypochlorite. We characterize different C. difficile clinical isolates and assess the chemical’s impact on spores’ biochemical composition. Changes in the biochemical composition can, in turn, change spores’ vibrational spectroscopic fingerprints, which can impact the possibility of detecting spores in a hospital using Raman based methods. Results: We found that the isolates show significantly different susceptibility to hypochlorite, with the R20291 strain, in particular, showing less than 1 log reduction in viability for a 0.5% hypochlorite treatment, far below typically reported values for C. difficile. While TEM and Raman spectra analysis of hypochlorite-treated spores revealed that some hypochlorite-exposed spores remained intact and not distinguishable from controls, most spores showed structural changes. These changes were prominent in B. thuringiensis spores than C. difficile spores. Conclusion: This study highlights the ability of certain C. difficile spores to survive practical disinfection exposure and the related changes in spore Raman spectra that can be seen after exposure. These findings are important to consider when designing practical disinfection protocols and vibrational-based detection methods to avoid a false-positive response when screening decontaminated areas. Graphical

Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systematic approach was used to increase glycoprotein production titres of an exemplar protein, AcrA, and the resulting glycosylation efficiency was quantified using a combination of Western blots and pseudo Selective Reaction Monitoring (pSRM). Western blot and pSRM results demonstrate that codon optimising the oligosaccharyltransferase, PglB, for E. coli expression, increases efficiency by 77% and 101%, respectively. Furthermore, increasing expression of glycosyltransferase, WecA, in E. coli improves efficiency by 43% and 27%, respectively. However, increasing the amount of donor lipid used in the glycosylation process did not impact on the glycosylation efficiency in this system, with this specific protein.

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.

Microwaves are a form of non-ionizing radiation composed of electric (E) and magnetic (H) fields and are absorbed by biological tissues with a high water content. Our study investigated the effect of the E field, H field, and a combination of both (E + H) field’s exposure of structurally diverse micro-organisms, at a frequency of 2.45 GHz. We observed that the exposure to a microwave E field of an amplitude of 9.3 kV/m had no significant effect on cell viability; however, it did increase membrane permeability of Mycobacterium smegmatis to propidium iodide and to a range of different sized dextran particles in Escherichia coli, Staphylococcus aureus, Candida albicans, and M. smegmatis. The permeability of propidium iodide was observed in microwave treated cells (M. smegmatis) but not in heat-treated cells. Permeability of 3 kDa sized fluorescently labeled dextrans was observed across all cell types; however, this was found not to be the case for larger 70 kDa dextran particles. In terms of efflux, DNA was detected following E field exposure of M. smegmatis. In contrast, H field exposure had no effect on cell viability and did not contribute to increase cell’s membrane to dextran particles. In conclusion, this study shows that microwave generated E fields can temporarily disrupt membrane integrity without detrimentally impacting on cell viability. This approach has the potential to be developed as a high efficiency electropermeabilization method and as a means of releasing host DNA to support diagnostic applications.

Bacterial spores are highly resistant to heat, radiation and various disinfection chemicals. The impact of these on the biophysical and physicochemical properties of spores can be studied on the single‐cell level using optical tweezers. However, the effect of the trapping laser on spores' germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose‐response relationship, with only 15% of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65%. The results thereby indicate that molecular oxygen is a major contributor to the germination‐inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.