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The utility of anaerobic blood culture bottles remains controversial, especially for specimens from children. Data are limited on the inclusion of an anaerobic bottle as part of a blood culture “set” when using contemporary blood culture instruments and media. Here, we evaluated the clinical utility of anaerobic blood culture bottles (FN Plus) and aerobic bottles (FA Plus) for the BacT/Alert Virtuo blood culture system (bioMérieux). A total of 158,710 bottles collected between November 2018 and October 2019 were evaluated. There were 6,652 positive anaerobic bottles, of which 384 (5.8%) contained 403 obligate anaerobes. In patients

We report the draft genome sequence of an unusual Mycobacterium isolate recovered from a patient’s arm tissue. The 4,025,753-bp draft genome exhibits a GC content of 71.02%, and a 16S rRNA gene analysis found that the closest relative was Mycobacterium grossiae .

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 being discarded as negative has remained 5 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., 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 being discarded as negative has remained 5 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 institutional review board (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 and 108 h, respectively. The mean (median) times to positivity were 18.4 h (15.6 h) for Staphylococcus aureus, 12.3 h (9.5 h) for Escherichia coli, 22.2 h (15.9 h) for Pseudomonas aeruginosa, and 48.9 h (42.9 h) for Candida spp. Only 175 bottles (0.1% of all bottles) flagged positive after 4 days of incubation; 89 (51%) of these bottles grew Cutibacterium (Propionibacterium) species. Chart review of blood cultures positive after 4 days (96 h) rarely had a clinical impact and sometimes had a negative impact on patient care. Finally, a seeded study of the HACEK group (i.e., Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella), historically associated with delayed blood culture positivity, demonstrated no benefit to extended incubation beyond 4 days. Collectively, these findings demonstrated that a 4-day incubation time was sufficient for the Virtuo system and media. Implementation of the 4-day incubation time could enhance clinically relevant results by reducing recovery of contaminants and finalizing blood cultures 1 day earlier.

Clinical and public health laboratories throughout the world have rapidly expanded diagnostic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1-3).…

Aztreonam-avibactam is a combination antimicrobial agent with activity against carbapenemase-producing Enterobacteriaceae (CPE) with metallo-β-lactamases (MβLs). Although aztreonam-avibactam is not yet approved by the U.S. Food and Drug Administration (FDA), clinicians can administer this combination by using two FDA-approved drugs: aztreonam and ceftazidime-avibactam. This combination of drugs is recommended by multiple experts for treatment of serious infections caused by MβL-producing CPE. At present, in vitro antimicrobial susceptibility testing (AST) of aztreonam-avibactam is not commercially available; thus, most clinicians receive no laboratory-based guidance that can support consideration of aztreonam-avibactam for serious CPE infections. Here, we report our internal validation for aztreonam-avibactam AST by reference broth microdilution (BMD) according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The validation was performed using custom frozen reference BMD panels prepared in-house at the Centers for Disease Control and Prevention (CDC). In addition, we took this opportunity to evaluate a new panel-making method using a digital dispenser, the Hewlett Packard (HP) D300e. Our studies demonstrate that the performance characteristics of digitally dispensed panels were equivalent to those of conventionally prepared frozen reference BMD panels for a number of drugs, including aztreonam-avibactam. We found the HP D300e digital dispenser to be easy to use and to provide the capacity to prepare complex drug panels. Our findings will help other clinical and public health laboratories implement susceptibility testing for aztreonam-avibactam.

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 σ v and that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.

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 m idcell l ocalizing d ivision 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.