Routine Use of Anaerobic Blood Culture Bottles for Specimens Collected from Adults and Children Enhances Microorganism Recovery and Improves Time to Positivity.

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 <19 years old, there were 389 positive anaerobic bottles, with 15 (1.8%) containing 16 obligate anaerobes. If not for anaerobic bottles, all but 8 obligate anaerobes would have gone undetected. Furthermore, anaerobic bottles were advantageous for some facultative anaerobes. Staphylococcus aureus from anaerobic bottles demonstrated statistically significant increased recovery (1,992 anaerobic versus 1,901 aerobic bottles, P = 0.009) and faster mean time to positivity (1,138 versus 1,174 min, P = 0.027). Only 25 microorganisms had statistically significant improved recovery and/or faster time to positivity from aerobic versus anaerobic bottles, suggesting anaerobic bottles offer comparable growth for most species. Finally, if only an aerobic bottle had been collected, 2,027 fewer positive cultures would have been detected and 7,452 fewer isolates would have been reported, including cultures with S. aureus (413 isolates, 10.6% less), Pseudomonas aeruginosa (9 isolates, 3.1% less) and Escherichia coli (193 isolates, 14.0% less). Taken together, these findings support the practice of routinely including an anaerobic bottle for blood culture collection.

Draft Genome Sequence of a Mycobacterium Strain Isolated from a Clinical Wound Sample.

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.

Deep Convolutional Neural Networks Implementation for the Analysis of Urine Culture.

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.

Comparative Genomics of Borderline Oxacillin-Resistant Staphylococcus aureus Detected during a Pseudo-outbreak of Methicillin-Resistant S. aureus in a Neonatal Intensive Care Unit.

Active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) is a component of our neonatal intensive care unit (NICU) infection prevention efforts. Recent atypical trends prompted review of 42 suspected MRSA isolates. Species identification was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and methicillin resistance was reevaluated by PBP2a lateral flow assay, cefoxitin/oxacillin susceptibility testing, mecA and mecC PCR, and six commercially available MRSA detection agars. All isolates were confirmed S. aureus, but only eight were MRSA (cefoxitin resistant, PBP2a positive, mecA positive, growth on all MRSA screening agars). One MRSA isolate was cefoxitin susceptible but PBP2a and mecA positive, and the remaining 33 were cefoxitin susceptible, PBP2a negative, and mecA negative; interestingly, these isolates grew inconsistently across MRSA screening agars and had susceptibility profiles consistent with that of borderline oxacillin-resistant S. aureus (BORSA). Comparative genomic analyses found these BORSA isolates to be phylogenetically diverse and not representative of clonal expansion or shared gene content, though clones of two NICU strains were infrequently observed over 8 months. We identified 6 features-substitutions and truncations in PBP2, PBP4, and GdpP and beta-lactamase hyperproduction-that were used to generate a random forest classifier to distinguish BORSA from methicillin-susceptible S. aureus (MSSA) in our cohort. Our model demonstrated a robust ability to predict the BORSA phenotype among isolates collected across two continents (validation area under the curve [AUC], 0.902). Taking these findings together, we observed an unexpected prevalence of BORSA in our NICU, BORSA misclassification by existing MRSA screening methods, and markers that are together discriminatory for BORSA and MSSA within our cohort. This work has implications for epidemiological reporting of MRSA rates for centers using different screening methods. IMPORTANCE In this study, we found a high prevalence of Staphylococcus aureus isolates exhibiting a borderline oxacillin resistance phenotype (BORSA) in our neonatal intensive care unit (NICU) serendipitously due to the type of MRSA screening agar used by our laboratory for active surveillance cultures. Subsequent phenotypic and molecular characterization highlighted an unexpected prevalence and variability of BORSA isolates. Through whole-genome sequencing, we interrogated core and accessory genome content and generated a random forest classification model to identify mutations and truncations in the PBP2, PBP4, and GdpP proteins and beta-lactamase hyperproduction, which correlated with BORSA and MSSA phenotypes among S. aureus clinical isolates collected across two continents. In consideration of these findings, this work will help clinical microbiology laboratories and clinicians identify MRSA screening shortfalls and draw attention to the non-mecA-mediated BORSA phenotype.

Multiplatform Assessment of Saliva for SARS-CoV-2 Molecular Detection in Symptomatic Healthcare Personnel and Patients Presenting to the Emergency Department.

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 < 0.0001). We found a 76.47% (26/34) PPA for Simplexa COVID-19 Direct Kit on saliva and a 67.6% (23/34) PPA for SalivaDirect compared to NP swab results. CONCLUSION: These data demonstrate molecular assays have variability in performance for detection of SARS-CoV-2 in saliva.

In Vitro Activity of Novel Antifungal Olorofim against Filamentous Fungi and Comparison to Eight Other Antifungal Agents.

Olorofim is a novel antifungal drug that belongs to the orotomide drug class which inhibits fungal dihydroorotate dehydrogenase (DHODH), thus halting pyrimidine biosynthesis and ultimately DNA synthesis, cell growth and division. It is being developed at a time when many invasive fungal infections exhibit antifungal resistance or have limited treatment options. The goal of this study was to evaluate the in vitro effectiveness of olorofim against a large collection of recently isolated, clinically relevant American mold isolates. In vitro antifungal activity was determined for 246 azole-susceptible Aspergillus fumigatus isolates, five A. fumigatus with TR34/L98H-mediated resistance, 19 Rhizopus species isolates, 21 Fusarium species isolates, and one isolate each of six other species of molds. Olorofim minimum inhibitory concentrations (MICs) were compared to antifungal susceptibility testing profiles for amphotericin B, anidulafungin, caspofungin, isavuconazole, itraconazole, micafungin, posaconazole, and voriconazole. Olorofim MICs were significantly lower than those of the echinocandin and azole drug classes and amphotericin B. A. fumigatus wild type and resistant isolates shared the same MIC50 = 0.008 µg/mL. In non-Aspergillus susceptible isolates (MIC ≤ 2 µg/mL), the geometric mean (GM) MIC to olorofim was 0.54 µg/mL with a range of 0.015-2 µg/mL. Olorofim had no antifungal activity (MIC ≥ 2 µg/mL) against 10% of the collection (31 in 297), including some isolates from Rhizopus spp. and Fusarium spp. Olorofim showed promising activity against A. fumigatus and other molds regardless of acquired azole resistance.

IL-7 Immunotherapy in a Nonimmunocompromised Patient With Intractable Fungal Wound Sepsis.

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.

Staphylococcus aureus injection drug use-associated bloodstream infections are propagated by community outbreaks of diverse lineages.

Background: 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). Methods: 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. Results: 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. Conclusions: 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.

Evaluation of Infectious Disease Test Ordering and Positivity Rates in Illicit Fentanyl Users.

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.

Assessing the in vitro impact of ceftazidime on aztreonam/avibactam susceptibility testing for highly resistant MBL-producing Enterobacterales.

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.

Evaluation of Optimal Blood Culture Incubation Time To Maximize Clinically Relevant Results from a Contemporary Blood Culture Instrument and Media System.

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.

Genomic Prediction of Antimicrobial Resistance: Ready or Not, Here It Comes!

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.

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile.

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, σTcdR , 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 σTcdR 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, σD and CodY, revealed neither is required for bimodal toxin gene expression. However, σD 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 σTcdR , 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.

Use of mCherryOpt Fluorescent Protein in Clostridium difficile.

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.

Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile.

Fluorescent proteins are powerful reporters in biology, but most require O2 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 CFPopt 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 CFPopt. 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.

Identification and characterization of a gene cluster required for proper rod shape, cell division, and pathogenesis in Clostridium difficile.

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 O2-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.

Interspecies signaling between Veillonella atypica and Streptococcus gordonii requires the transcription factor CcpA.

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 alpha-amylase. We show that the transcription factor CcpA is required for this interspecies interaction.