Jeffrey M. Kubiak

Objective: To reduce the use of follow-up blood cultures (FUBC), that is repeat blood cultures (BCs) used to monitor patients for clearance of a prior positive BC, or with new/worsening symptoms after negative BCs, by 10%. Design: Multicenter quality improvement study. Setting: A network of 8 hospitals, including academic tertiary care centers and community hospitals. Participants: 40,021 patients, including adult, pediatric, inpatients, and outpatients, for whom blood cultures were ordered over a 24-week period in 2024. Interventions: We implemented a series of iterative Plan-Do-Study-Act (PDSA) cycles to guide clinicians toward appropriate BC ordering. These cycles included educational memos, limiting the ability to order more than one BC at a time, a best practice advisory in the electronic health record to limit BC orders with a prior negative within 72 hours, and a new order panel for BCs that defaulted FUBC to a single set. Results: Prior to the intervention period, FUBCs comprised 29% of all BC orders placed. After the intervention, there was an 18% absolute reduction in FUBC, primarily due to decreased use of paired FUBC sets. There were no increases in the length of stay, in-hospital mortality, or 30-day readmission. Conclusions: We exceeded our goal to decrease FUBCs using diagnostic stewardship. This approach to FUBCs is sustainable and scalable as part of diagnostic stewardship strategies for BC stewardship.

Background: Lawsonella clevelandensis is a recently characterized fastidious anaerobic Gram-positive, partially acid-fast bacillus associated with monomicrobial abscess formation. Case Summary: This case report features a 56-year-old female who developed right breast pain and was found to have subareolar breast abscess. Aspiration of the abscess grew pinpoint to small waxy colonies within 5 days on phenylethyl alcohol agar under anaerobic conditions. The organism was maintained with sustained growth from a subculture in thioglycolate broth. Conventional identification techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, failed to identify the organism, which was confirmed as L. clevelandensis by 16S rDNA sequencing. Conclusion: This case marks the fifth reported case of L. clevelandensis as the causative organism of monomicrobial breast abscess. Additionally, this case marks the first reported instance of L. clevelandensis being maintained and cultivated within liquid-based growth media.

Background: Creutzfeldt–Jakob disease (CJD) is a rare, fatal, and transmissible neurodegenerative disorder caused by prion proteins. Handling specimens from individuals with suspected or confirmed cases presents a safety challenge to hospital workers including clinical laboratory staff. As no national guidelines exist, the clinical pathology laboratory must establish protocols for handling these specimens to ensure sufficient protective measures. This study aims to explore how various medical institutions manage CJD specimens, as a first step toward developing standardized preanalytical protocols for safe specimen handling by health care professionals. Methods: An electronic survey was generated and disseminated to diplomats of the American Board of Clinical Chemistry and was posted on the Listserv platform of the American Society for Microbiology and the Artery forum of the Association for Diagnostics and Laboratory Medicine. The survey evaluated various procedures and precautions implemented, the nature of the specimens processed, and whether they are processed in-house or sent to reference laboratories. Results: A total of 49 responses were collected. Most respondents (64%) noted their laboratories process specimens with a clinical suspicion of CJD regardless of the level of suspicion, 13% handled specimens only if the degree of suspicion was low, and 16% did not process specimens in-house at all. Among those who process CJD specimens, practices varied greatly, including different levels of precautions, use of biological safety cabinets, aliquoting, disposal, and disinfection procedures. Conclusions: A lack of standardization across laboratories exists for the handling of specimens of patients with suspected CJD. This study summarizes the approaches reported by survey respondents, providing a rationale for developing protocols for the safe handling of these specimens and highlighting the need to develop uniform universal standardized processing procedures.

Universal PCR for bacteria, mycobacteria, and fungi can aid in the diagnosis of occult infections, especially in the case of fastidious organisms or when prior antimicrobial treatment compromises culture growth. However, the limitations of this technology, including lack of specificity, high cost, long turnaround time, and lack of susceptibility data, may limit its effect on clinical outcomes. We performed a retrospective analysis of all specimens sent for universal PCR over a 10-year period from 2013 to 2022, focusing on clinical indications for test utilization and patient outcomes. All specimens required approval by a microbiology laboratory director prior to testing. A total of 708 specimens were sent from 638 patients. Of these specimens, 163 were positive, with an overall positivity rate of 23%. Pre-analytic factors associated with a positive universal PCR result were the presence of organisms on Gram stain or histology, the presence of neutrophils on Gram stain, and growth on culture. Positivity rates varied significantly by specimen type. A total of 20% of all organisms detected were deemed clinically irrelevant by the clinical services. A positive universal PCR led to a change in antibiotic management in 29% of cases. Positive fungal universal PCR results sent from hospitalized patients were associated with worse outcomes, including increased hospital mortality. Our findings suggest that factors such as the presence of organisms or neutrophils on Gram stain, specimen source/clinical context, and anticipated changes in management based on results should be strongly considered when making stewardship decisions regarding the appropriateness of this testing modality. IMPORTANCE: Our work provides a retrospective analysis of universal PCR orders for bacteria, mycobacteria, and fungi across our institution across a 10-year period. We assessed the positivity rates for this diagnostic tool by test type and specimen type and, critically, studied whether and how the results influenced the outcomes from treatment change, to readmission, to death.

Universal PCR for bacteria, mycobacteria, and fungi can aid in the diagnosis of occult infections, especially in the case of fastidious organisms or when prior antimicrobial treatment compromises culture growth. However, the limitations of this technology, including lack of specificity, high cost, long turnaround time, and lack of susceptibility data, may limit its effect on clinical outcomes. We performed a retrospective analysis of all specimens sent for universal PCR over a 10-year period from 2013-2022, focusing on clinical indications for test utilization and patient outcomes. A total of 708 specimens were sent from 638 patients. Of those specimens, 163 were positive, for an overall positivity rate of 23%. Pre-analytic factors associated with a positive universal PCR result were the presence of organisms on Gram stain or histology, the presence of neutrophils on Gram stain, and growth on culture. Positivity rates varied significantly by specimen type. 20% of all organisms detected were deemed contaminants by the clinical services. A positive universal PCR led to a change in antibiotic management in 29% of cases. Positive fungal universal PCR results sent from hospitalized patients were associated with worse outcomes, including increased hospital mortality. Our findings suggest that factors such as the presence of organisms or neutrophils on Gram stain, specimen source/clinical context, and anticipated changes in management based on results should be strongly considered when making stewardship decisions regarding the appropriateness of this testing modality.

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter’s activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of ‘early’, ‘middle’, and ‘late’ genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene’s transcription as a function of stimulus dose. As the precise timing of gene expression is critical for cells to respond and adapt to new environments, it is important to understand the underlying mechanisms which control this timing. In this report, we studied the timing of transcription for genes in the bacterial DNA damage repair pathway (known as the SOS response), a regulatory system where each gene is controlled by the same transcriptional repressor, LexA. By specifically isolating the role of the LexA binding interaction at SOS gene promoters, we found a relationship between the amount of DNA damage incurred by the cell, LexA binding kinetics at a promoter, and the timing of promoter activation. Our data fit a kinetic model that reveals how a disequilibrium between the LexA-operator binding reaction and cellular LexA concentrations causes timing differences between genes to emerge only at higher doses of DNA damage. Taken together, we show that non-equilibrium DNA binding kinetics is the mechanism by which a single transcription factor can modulate timing differences across an entire network of genes as a function of stimulus dose.

The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.