Aims The objective of this study was to determine the effects of Ca‐dipicolinic acid (CaDPA), cortex‐lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. Methods and Results Bacillus subtilis spores, wild‐type, CaDPA‐less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine‐treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild‐type and CaDPA‐less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores ± CaDPA, with up to 99% killing with 1 mol l−1 dodecylamine in 4 h at 45°C with spores at ~108 ml−1. Dodecylamine killing of decoated wild type and CLE‐less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA‐less spores, with ≥99% killing in 5 min. Propidium iodide stained intact spores ± CaDPA minimally, decoated CaDPA‐replete spores or dodecylamine‐killed CLE‐less spores peripherally, and cores of decoated CaDPA‐less spores and dodecylamine‐killed intact spores with CLEs. The IM of some decoated CaDPA‐less spores was greatly reorganized. Conclusions Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. Significance and Impact of the Study This work gives new information on killing bacterial spores by dodecylamine, and how spore IM’s relative impermeability is maintained.

Bacteria have a repertoire of strategies to overcome antibiotics in clinical use, complicating our ability to treat and cure infectious diseases. In addition to evolving resistance, bacteria within genetically clonal cultures can undergo transient phenotypic changes and tolerate high doses of antibiotics. These cells, termed persisters, exhibit heterogeneous phenotypes: the strategies that a bacterial population deploys to overcome one class of antibiotics can be distinct from those needed to survive treatment with drugs with another mode of action. It was previously reported that fluoroquinolones, which target DNA topoisomerases, retain the capacity to kill non-growing bacteria that tolerate other classes of antibiotics. Here, we show that in Escherichia coli stationary-phase cultures and colony biofilms, persisters that survive treatment with the anionic fluoroquinolone Delafloxacin depend on the AcrAB-TolC efflux pump. In contrast, we did not detect this dependence on AcrAB-TolC in E. coli persisters that survive treatment with three other fluroquinolone compounds. We found that the loss of AcrAB-TolC activity via genetic mutations or chemical inhibition not only reduces Delafloxacin persistence in non-growing E. coli MG1655 or EDL933 (an E. coli O157:H7 strain), it limits resistance development in progenies derived from Delafloxacin persisters that were given the opportunity to recover in nutritive media following antibiotic treatment. Our findings highlight the heterogeneity in defense mechanisms that persisters use to overcome different compounds within the same class of antibiotics. They further indicate that efflux pump inhibitors can potentiate the activity of Delafloxacin against stationary-phase E. coli and block resistance development in Delafloxacin persister progenies.

Antibiotic persistence is a phenomenon in which rare cells of a clonal bacterial population can survive antibiotic doses that kill their kin, even though the entire population is genetically susceptible. With antibiotic treatment failure on the rise, there is growing interest in understanding the molecular mechanisms underlying bacterial phenotypic heterogeneity and antibiotic persistence. However, elucidating these rare cell states can be technically challenging. The advent of single-cell techniques has enabled us to observe and quantitatively investigate individual cells in complex, phenotypically heterogeneous populations. In this review, we will discuss current technologies for studying persister phenotypes, including fluorescent tags and biosensors used to elucidate cellular processes; advances in flow cytometry, mass spectrometry, Raman spectroscopy, and microfluidics that contribute high-throughput and high-content information; and next-generation sequencing for powerful insights into genetic and transcriptomic programs. We will further discuss existing knowledge gaps, cutting-edge technologies that can address them, and how advances in single-cell microbiology can potentially improve infectious disease treatment outcomes.

Spores of Firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple Firmicute species and relative mRNA levels determined by RNA-seq. Determination of RNA levels in single spores allowed calculation of RNA nt/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at > 1/spore. Bacillus subtilis, Bacillus atrophaeus and Clostridiodes difficile spores had 49, 42 and 51 mRNAs at > 1/spore, numbers of mRNAs at ≥ 1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores, and ∼4-fold higher in Bacillus megaterium spores. In all species some to many abundant spore mRNAs: i) were transcribed by RNA polymerase with forespore-specific σ factors; ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores; and iii) a few were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by RT-qPCR analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all Firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. Importance Only ∼6% of mRNAs in spores of six Firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that Firmicute spores’ abundant mRNAs are not translated when spores germinate, but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

Antibiotic tolerant bacteria and persistent cells that remain alive after a course of antibiotic treatment can foster the chronicity of infections and the development of antibiotic resistance. Elucidating how bacteria overcome antibiotic action and devising strategies to bolster a new drug’s activity can allow us to preserve our antibiotic arsenal.