Fluorescent proteins are powerful reporters in biology, but most require O 2 for chromophore maturation, making them inherently difficult to use in anaerobic bacteria. Clostridium difficile, a strict anaerobe with a genomic GC content of only 29%, is the leading cause of hospital-acquired diarrhea in developed countries, and new methods for studying this pathogen are sorely needed. We recently demonstrated that a cyan fluorescent protein called CFP opt that has been codon optimized for production in low-GC bacteria can be used to study protein localization in C. difficile provided the cells are fixed prior to exposure to air. We describe here a codon-optimized variant of mCherry (mCherryOpt) that exhibits faster acquisition of fluorescence and a better signal-to-noise ratio than CFP opt . We utilized mCherryOpt to construct plasmids for studying protein localization (pRAN473) and gene expression (pDSW1728) in C. difficile. Plasmid pRAN473 is an mCherryOpt fusion vector with a tetracycline-inducible promoter. To document its biological utility, we demonstrated septal localization of two cell division proteins, MldA and ZapA. Plasmid pDSW1728 is designed for cloning a promoter of interest upstream of mCherryOpt. As proof of principle, we studied the expression of the pdaV operon, which is required for lysozyme resistance. In confirmation and extension of previous reports, we found that expression of the pdaV operon requires the alternative sigma factor v and that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.C lostridium difficile is a low-GC, spore-forming bacterium that is burdening the health care systems of developed countries (1-3). While genetic techniques to study C. difficile are becoming increasingly available, the repertoire of tools remains limited. This is due in part to the strictly anaerobic environment required to grow C. difficile.Green fluorescent protein (GFP) can be produced in cells grown anaerobically, but it is unable to fluoresce because chromophore maturation requires O 2 for dehydration reactions that introduce double bonds into amino acids (4). Nevertheless, GFP produced in an anaerobic environment can mature and fluoresce upon subsequent exposure to air (4, 5). We recently took advantage of this observation to show that GFP can be used to localize cell division proteins in anaerobically grown Escherichia coli (6). Similarly, we showed that a derivative of cyan fluorescent protein named CFP opt (because it has been codon optimized for low-GC bacteria) can be used to localize cell division proteins in anaerobically grown C. difficile (6). In both organisms, it was necessary to fix cells anaerobically to preserve their architecture and then expose them to air overnight to allow chromophore maturation, which required many hours. Fixation was necessary in the case of E. coli to ensure that the localization observed reflected anaerobic conditions rather than subsequent adaptation to air. In the case of C. difficile, fixation was necessary b...

Little is known about cell division in Clostridium difficile, a strict anaerobe that causes serious diarrheal diseases in people whose normal intestinal microbiome has been perturbed by treatment with broad-spectrum antibiotics. Here we identify and characterize a gene cluster encoding three cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, but MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of cyan fluorescent protein (CFP) to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. This application of CFP was possible because we discovered that O 2 -dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they might be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome.C lostridium difficile is a strictly anaerobic, Gram-positive, spore-forming bacterium that has become the leading cause of hospital-acquired diarrhea in developed countries. The annual impact of C. difficile infections in the United States has been estimated at 14,000 deaths and over $1 billion in excess medical costs (1). Both the severity and the frequency of C. difficile infections are increasing (2), and a recent report on the impact of antibiotic resistance classified the organism as an "urgent threat," the highest threat level (1).C. difficile infections typically occur in people who have been treated with antibiotics that disrupt the flora of the gastrointestinal tract (3, 4). Although C. difficile is resistant to many antibiotics, the infection usually resolves upon treatment with metronidazole or oral vancomycin (5). Unfortunately, disease recurs in ϳ20% of patients, and the prognosis for this cohort is poor (4, 6, 7). The high rate of recurrence has been attributed to germination of C. difficile spores after antibiotic therapy is ended but before restoration of the normal flora (2, 8). For this reason, there is interest in developing antibiotics that target C. difficile selectively and in treatments such as fecal transplants which work by restoring a healthy microbiome (4, 7, 9, 10).Here we describe a cluster of three genes found in C. difficile that is important for morphogenesis, cell division, and pathogenesis. W...

Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σ , which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σ production by engineering cells to express tcdR from a tetracycline-inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σ and CodY, revealed neither is required for bimodal toxin gene expression. However, σ biased cells toward the Toxin-ON state, while CodY biased cells toward the Toxin-OFF state. Finally, toxin gene expression was observed in sporulating cells. We conclude that (i) toxin production is regulated by a bistable switch governed by σ , which only accumulates to high enough levels to trigger toxin gene expression in a subset of cells, and (ii) toxin production and sporulation are not mutually exclusive developmental programs.

Here we describe protocols for using the red fluorescent protein mCherryOpt in Clostridium difficile. The protocols can be readily adapted to similar fluorescent proteins (FPs), such as green fluorescent protein (GFP) and cyan fluorescent protein (CFP). There are three critical considerations for using FPs in C. difficile. (1) Choosing the right color: Blue and (especially) red are preferred because C. difficile exhibits considerable yellow-green autofluorescence. (2) Codon optimization: Most FP genes in general circulation have a GC content of ~60 %, so they are not well expressed in low-GC bacteria. (3) Fixing anaerobically grown cells prior to exposure to O2: The FPs under consideration here are non-fluorescent when produced anaerobically because O2 is required to introduce double bonds into the chromophore. Fixation prevents C. difficile cells from becoming degraded during the several hours required for chromophore maturation after cells are exposed to air. Fixation can probably be omitted for studies in which maintaining cellular architecture is not important, such as using mCherryOpt to monitor gene expression.

Timely diagnosis of microorganisms in blood cultures is necessary to optimize therapy. Although blood culture media and systems have evolved for decades, the standard interval for incubation prior to discard as negative has remained five days. Here, we evaluated the optimal incubation time for the BACT/ALERT VIRTUO blood culture detection system (bioMérieux) using FA Plus (aerobic) and FN Plus (anaerobic) resin culture bottles in routine clinical use. Following IRB approval, a retrospective review evaluated the outcomes of 158,710 bottles collected between November 2018 and October 2019. The number of positive blood bottles was 13,592 (8.6%); 99% of positive aerobic and anaerobic bottles flagged positive by 91.5 h and 108 h, respectively. The mean (median) time-to-positivity for Staphylococcus aureus was 18.4 h (15.6 h), Escherichia coli 12.3 h (9.5 h), Pseudomonas aeruginosa 22.2 h (15.9 h), and Candida spp. 48.9 h (42.9 h). Only 175 bottles (0.1% of all bottles) flagged positive after four days of incubation; 89 (51%) of these bottles grew Cutibacterium (Propionibacterium) species. Chart review of blood cultures positive after four days (96 h) rarely had clinical impact, and sometimes had a negative impact on patientcare. Finally, a seeded study of the HACEK group, historically associated with delayed blood culture positivity, demonstrated no benefit to extended incubation beyond four days. Collectively, these findings demonstrated that a four-day incubation time was sufficient for the VIRTUO system and media. Implementation of the four-day incubation time could enhance clinically relevant results by reducing recovery of contaminants and finalizing blood cultures one day earlier.