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Background Urine culture images collected using bacteriology automation are currently interpreted by technologists during routine standard-of-care workflows. Machine learning may be able to improve the harmonization of and assist with these interpretations. Methods A deep learning model, BacterioSight, was developed, trained, and tested on standard BD-Kiestra images of routine blood agar urine cultures from 2 different medical centers. Results BacterioSight displayed performance on par with standard-of-care–trained technologist interpretations. BacterioSight accuracy ranged from 97% when compared to standard-of-care (single technologist) and reached 100% when compared to a consensus reached by a group of technologists (gold standard in this study). Variability in image interpretation by trained technologists was identified and annotation “fuzziness” was quantified and found to correlate with reduced confidence in BacterioSight interpretation. Intra-testing (training and testing performed within the same institution) performed well giving Area Under the Curve (AUC) ≥0.98 for negative and positive plates, whereas, cross-testing on images (trained on one institution’s images and tested on images from another institution) showed decreased performance with AUC ≥0.90 for negative and positive plates. Conclusions Our study provides a roadmap on how BacterioSight or similar deep learning prototypes may be implemented to screen for microbial growth, flag difficult cases for multi-personnel review, or auto-verify a subset of cultures with high confidence. In addition, our results highlight image interpretation variability by trained technologist within an institution and globally across institutions. We propose a model in which deep learning can enhance patient care by identifying inherent sample annotation variability and improving personnel training.

The ongoing injection drug use (IDU) crisis in the United States has been complicated by an emerging epidemic of Staphylococcus aureus IDU-associated bloodstream infections (IDU-BSI). We performed a case-control study comparing S. aureus IDU-BSI and non-IDU BSI cases identified in a large US Midwestern academic medical center between Jan 1, 2016 and Dec 21, 2019. We obtained the whole-genome sequences of 154 S. aureus IDU-BSI and 91 S. aureus non-IDU BSI cases, which were matched with clinical data. We performed phylogenetic and comparative genomic analyses to investigate clonal expansion of lineages and molecular features characteristic of IDU-BSI isolates. Here we show that patients with IDU-BSI experience longer durations of bacteremia and have lower medical therapy completion rates. In phylogenetic analyses, 45/154 and 1/91 contemporaneous IDU-BSI and non-IDU BSI staphylococcal isolates, respectively, group into multiple, unique clonal clusters, revealing that pathogen community transmission distinctively spurs IDU-BSI. Lastly, multiple S. aureus lineages deficient in canonical virulence genes are overrepresented among IDU-BSI, which may contribute to the distinguishable clinical presentation of IDU-BSI cases. We identify clonal expansion of multiple S. aureus lineages among IDU-BSI isolates, but not non-IDU BSI isolates, in a community with limited access to needle exchange facilities. In the setting of expanding numbers of staphylococcal IDU-BSI cases consideration should be given to treating IDU-associated invasive staphylococcal infections as a communicable disease. Marks and Calix et al. perform a case-control genomic epidemiology study comparing Staphylococcus aureus isolates from intravenous drug use-associated bloodstream infections with those from other bloodstream infections. The authors show there is clonal expansion of lineages in intravenous drug use-associated bloodstream infections suggesting person-to-person transmission of S. aureus. Persons who inject drugs are at increased risk of developing a bloodstream infection caused by the bacterium Staphylococcus aureus. To investigate whether this risk is due to transmission of the bacterium within this community, we compared the complete set of genes (genome) of S. aureus isolated from people with bloodstream infections who do and do not inject drugs. S. aureus causing bloodstream infections in persons who inject drugs were much more likely to belong to one of multiple networks of very closely related subtypes, demonstrating that in some communities the bacteria causing this type of infection can likely be directly transmitted from person to person through high-risk injection drug use practices, such as sharing needles. Therefore, invasive infections in persons who inject drugs can spread like communicable diseases and this can inform future policy on how to prevent them.

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 .

Background Aztreonam/avibactam is a combination agent that shows promise in treating infections caused by highly antibiotic-resistant MBL-producing Enterobacterales. This combination can be achieved by combining two FDA-approved drugs: ceftazidime/avibactam and aztreonam. It is unknown whether ceftazidime in the combination ceftazidime/aztreonam/avibactam has a synergistic or antagonistic effect on the in vitro activity of aztreonam/avibactam by significantly increasing or decreasing the MIC. Objectives To determine whether increasing ceftazidime concentrations affect the MICs of aztreonam/avibactam alone. Methods A custom 8 × 8 chequerboard broth microdilution (BMD) panel was made using a digital dispenser (Hewlett-Packard, Corvallis, OR, USA). The panel included orthogonal 2-fold dilution series of aztreonam and ceftazidime ranging from 0.5 to 64 mg/L. Avibactam concentration was kept constant at 4 mg/L throughout the chequerboard. Thirty-seven Enterobacterales isolates from the CDC & FDA Antibiotic Resistance Isolate Bank or CDC’s internal collection with intermediate or resistant interpretations to aztreonam and ceftazidime/avibactam were included for testing. All isolates harboured at least one of the following MBL genes: blaIMP, blaNDM or blaVIM. Results Regardless of the concentration of ceftazidime, aztreonam/avibactam with ceftazidime MICs for all 37 isolates were within one 2-fold doubling dilution of the aztreonam/avibactam MIC. Conclusions Ceftazidime, in the combination ceftazidime/avibactam/aztreonam, did not affect the in vitro activity of aztreonam/avibactam in this sample of isolates. These findings can help assure clinical and public health laboratories that testing of aztreonam/avibactam by BMD can act as a reliable surrogate test when the combination of ceftazidime/avibactam and aztreonam is being considered for treatment of highly antibiotic-resistant MBL-producing Enterobacterales.

Background Saliva has garnered great interest as an alternative specimen type for molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data are limited on the relative performance of different molecular methods using saliva specimens and the relative sensitivity of saliva to nasopharyngeal (NP) swabs. Methods To address the gap in knowledge, we enrolled symptomatic healthcare personnel (n = 250) from Barnes-Jewish Hospital/Washington University Medical Center and patients presenting to the Emergency Department with clinical symptoms compatible with coronavirus disease 2019 (COVID-19; n = 292). We collected paired saliva specimens and NP swabs. The Lyra SARS-CoV-2 assay (Quidel) was evaluated on paired saliva and NP samples. Subsequently we compared the Simplexa COVID-19 Direct Kit (Diasorin) and a modified SalivaDirect (Yale) assay on a subset of positive and negative saliva specimens. Results The positive percent agreement (PPA) between saliva and NP samples using the Lyra SARS-CoV-2 assay was 63.2%. Saliva samples had higher SARS-CoV-2 cycle threshold values compared to NP swabs (P Conclusion These data demonstrate molecular assays have variability in performance for detection of SARS-CoV-2 in saliva.

A nonimmunocompromised patient developed life-threatening soft tissue infection with Trichosporon asahii, Fusarium, and Saksenaea that progressed despite maximum antifungal therapies and aggressive debridement. Interleukin-7 immunotherapy resulted in clinical improvement, fungal clearance, reversal of lymphopenia, and improved T-cell function. Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening fungal infections.

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).…

Background The emergence of illicit fentanyl use has resulted in considerable morbidity and mortality. Although illicit use of other opioids has been associated with transmission of viral and bacterial infections, limited data exist for the prevalence of infectious diseases among illicit fentanyl users. The purpose of this study was to assess the likelihood of infectious disease testing and infection prevalence among illicit fentanyl users. Methods Results from urine drug screens (UDSs) performed from August 13, 2019, to October 16, 2019, were obtained from the laboratory information system with concurrent microbial testing. Patients were categorized based on UDS results, and illicit drug use was inferred from physician encounter notes in the electronic medical record. Results Suspected illicit drugs users with fentanyl detected by UDS were more likely to be screened [odds ratio (OR): 1.7; 95% CI, 1.26–2.4] and test positive for hepatitis C virus (HCV) by immunoassay (OR: 5.89; 95% CI, 2.93–11.31) than patients without drugs detected. Patients with suspected illicit fentanyl use who were discharged from the emergency department (ED) were less likely to be tested for HCV than patients in outpatient settings (OR: 3.47; 95% CI, 1.05–10.4) and inpatient settings (OR: 17.43; 95% CI, 6.53–45.88). Patients with suspected illicit fentanyl use were more likely to have infected abscesses or wounds (OR: 5.12; 95% CI, 2.07–13.7) and Staphylococcus aureus infections (OR: 4.5; 95% CI, 1.59–12.28) than patients without drugs detected. Conclusions Patients with a positive UDS for fentanyl and suspected illicit use were more likely to test positive for HCV, were rarely screened for HCV in the ED, and had an increased risk of invasive S. aureus wound or abscess infection. These findings may represent considerable barriers to care for patients who use fentanyl illicitly.

Background Next-generation sequencing (NGS) technologies are being used to predict antimicrobial resistance. The field is evolving rapidly and transitioning out of the research setting into clinical use. Clinical laboratories are evaluating the accuracy and utility of genomic resistance prediction, including methods for NGS, downstream bioinformatic pipeline components, and the clinical settings in which this type of testing should be offered. Content We describe genomic sequencing as it pertains to predicting antimicrobial resistance in clinical isolates and samples. We elaborate on current methodologies and workflows to perform this testing and summarize the current state of genomic resistance prediction in clinical settings. To highlight this aspect, we include 3 medically relevant microorganism exemplars: Mycobacterium tuberculosis, Staphylococcus aureus, and Neisseria gonorrhoeae. Last, we discuss the future of genomic-based resistance detection in clinical microbiology laboratories. Summary Antimicrobial resistance prediction by genomic approaches is in its infancy for routine patient care. Genomic approaches have already added value to the current diagnostic testing landscape in specific circumstances and will play an increasingly important role in diagnostic microbiology. Future advancements will shorten turnaround time, reduce costs, and improve our analysis and interpretation of clinically actionable results.

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.

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(TcdR), 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(TcdR) 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(D) and CodY, revealed neither is required for bimodal toxin gene expression. However, σ(D) biased cells towards the Toxin-ON state, while CodY biased cells towards 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(TcdR), 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.

Background Azithromycin (AZI) is recommended with ceftriaxone (CRO) for treatment of uncomplicated gonococcal urethritis and cervicitis in the United States, and an AZI-susceptibility breakpoint is needed. Neither the Food and Drug Administration (FDA) nor the Clinical and Laboratory Standards Institute (CLSI) has set interpretive breakpoints for AZI susceptibility. As a result, AZI antimicrobial susceptibility testing (AST) cannot be interpreted using recognized standards. This has contributed to increasingly unavailable clinical laboratory AST, although gonorrhea is on the rise with >550 000 US gonorrhea cases reported to the Centers for Disease Control and Prevention in 2017, the highest number of cases since 1991. Methods This article summarizes the rationale data reviewed by the CLSI in June 2018. Results The CLSI decided to set a susceptible-only interpretive breakpoint at the minimum inhibitory concentration of ≤1 µg/mL. This is also the epidemiological cutoff value (ECV) (ie, the end of the wild-type susceptibility distribution). This breakpoint presumes that AZI (1-g single dose) is used in an approved regimen that includes an additional antimicrobial agent (ie, CRO 250 mg, intramuscular single dose). Conclusions Having a breakpoint can improve patient care and surveillance and allow future development and FDA regulatory approval of modernized AST to guide treatment. The breakpoint coincides with a European Committee on AST decision to remove previously established, differing AZI breakpoints and use the ECV as guidance for testing. The CLSI breakpoint is now the recognized standard that defines AZI susceptibility for gonococcal infections.

Background: Weekly surveillance to identify neonatal intensive care unit (NICU) infants with methicillin-resistant S. aureus (MRSA) nasal colonization was performed using Remel Spectra MRSA chromogenic media. An increased MRSA colonization rate from baseline was detected in 2019, prompting additional review of all positive MRSA NICU screening cultures from 2019. Methods: A subset of 23 positive cultures were interrogated in detail. Species-level identification was confirmed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) with a Bruker Biotyper. Penicillin-binding protein 2a (PBP2a) testing was performed using the Alere culture colony test, and cefoxitin and oxacillin susceptibility were assessed via Kirby-Bauer disk-diffusion methods (for the purpose of this analysis, oxacillin zone sizes 18 mm were considered susceptible). Molecular detection of mecA and mecC genes using PCR was performed. Results: All 23 isolates in the subset group were confirmed as S. aureus based on MALDI-TOF testing. Moreover, 8 isolates (35%) were confirmed as MRSA based on cefoxitin susceptibility, positive rapid PBP2a testing, and mecA PCR results. Overall, 15 isolates (65%) tested cefoxitin-susceptible and PBP2a negative with negative mecA and mecC gene testing. Of these, 1 (7%) tested oxacillin-susceptible based on disk-diffusion testing, consistent with methicillin-susceptible S. aureus (MSSA). The remaining 14 isolates (93%) tested oxacillin resistant based on oxacillin zone size. Conclusions: Our findings indicate the detection of mecA/mecC negative S. aureus isolates demonstrating oxacillin resistance and growth on Remel Spectra MRSA chromogenic media. These results have important implications for infection prevention surveillance efforts to detect MRSA and raise questions regarding optimal antibiotic therapy in patients with isolates displaying this phenotype.Funding: NoneDisclosures: None

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.

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.

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.

Streptococcus gordonii and Veillonella atypica , two early-colonizing members of the dental plaque biofilm, participate in a relationship that results in increased transcription of the S. gordonii gene amyB , encoding an α-amylase. We show that the transcription factor CcpA is required for this interspecies interaction.