Defining the value of medical microbiology consultation.

Medical microbiologists, defined as doctoral-level laboratory directors with subspecialty training in medical microbiology, lead the clinical laboratory operations through activities such as clinical consultations, oversight of diagnostic testing menu, institutional leadership, education, and scholastic activities. However, unlike their clinical colleagues, medical microbiologists are largely unable to bill for clinical consultations performed within the hospital and, therefore, unable to generate relative value units or a similar quantifiable metric. As hospital budgets tighten and justification of staffing becomes a necessity, this may present a challenge to the medical microbiologist attempting to prove their value to the organization. To aid in providing tangible data, the Personnel Standards and Workforce subcommittee of the American Society for Microbiology conducted a multi-center study across seven medical centers to document clinical consultations and their impact. Consults were generated equally from internal (laboratory-based) and external (hospital-based) parties, with the majority directly impacting patient management. Near universal acceptance of the medical microbiologist's recommendation highlights the worth derived from their expertise. External consults required more time commitment from the medical microbiologist than internal consults, although both presented ample opportunity for secondary value, including impact through stewardship, education, clinical guidance, and cost reduction. This study is a description of the content and impact of consultations that underscore the importance of the medical microbiologist as a key member of the healthcare team. IMPORTANCE: Medical microbiologists are invaluable to the clinical microbiology laboratory and the healthcare system as a whole. However, as medical microbiologists do not regularly generate relative value units, capturing and quantifying the value provided is challenging. As hospital budgets tighten, justification of staffing becomes a necessity. To aid in providing tangible data, the Personnel Standards and Workforce subcommittee of the American Society for Microbiology conducted a multi-center study across seven medical centers to document clinical consultations and their impact. To our knowledge, this is the first study to provide detailed evaluation of the consultative value provided by medical microbiologists.

Development and Validation of a Novel Anaerobic Carbapenem Inactivation Method (Ana-CIM) for the Detection of Carbapenemase Production in Bacteroides fragilis.

Antibiotic resistance, particularly to carbapenems, is of increasing concern in Bacteroides fragilis. Carbapenem resistance in B. fragilis is most often mediated by the activation of chromosomally encoded metallo-ß-lactamase cfiA by the presence of an upstream insertion sequence (IS). While traditional phenotypic susceptibility methods and molecular tests to detect carbapenem resistance in B. fragilis exist, they are not available in most clinical microbiology laboratory settings. Here, we describe the development of the anaerobic carbapenem inactivation method (Ana-CIM) for predicting carbapenemase production in B. fragilis based off the principles of the well-established modified carbapenem inactivation method (mCIM) for Enterobacterales and Pseudomonas aeruginosa. We also present the clinical validation and reproducibility of the Ana-CIM at three clinical laboratory sites (with 60 clinical isolates, 45% ertapenem resistant). Compared to ertapenem susceptibility by Etest interpreted by CLSI M100 Ed30, the Ana-CIM accurately detected carbapenem resistance in B. fragilis with categorical agreement (CA) of 87% (52/60) and 0% (0/21) very major error (VME), 11% (4/36) major error (ME), and 7% (4/60) minor error (mE) rates across all sites. Additionally, the Ana-CIM demonstrated high reproducibility with 5 clinical and 3 quality control (QC) isolates tested in triplicate with 3 commercial Mueller-Hinton media across all sites, with 93% (604/648) of replicates within a 2-mm zone size of the mode for each isolate. We conclude that the Ana-CIM can be readily deployed in clinical laboratories at a low cost for detection of carbapenemase-mediated resistance in B. fragilis.

A tale of two unusual anaerobic bacterial infections in an immunocompetent man: A case report and literature review.

We report a case of an immunocompetent man who presented with Desulfovibrio fairfieldensis bacteremia, followed by an epidural abscess due to Parvimonas micra. Only few cases have described unique clinical features related to both organisms, and this report illustrates two distinct sequential, if not concurrent, syndromes due to these anaerobes.

Evidence of Cross-Regulation in Two Closely Related Pyruvate-Sensing Systems in Uropathogenic Escherichia coli.

Two-component systems (TCSs) dictate many bacterial responses to environmental change via the activation of a membrane-embedded sensor kinase, which has molecular specificity for a cognate response regulator protein. However, although the majority of TCSs operate through seemingly strict cognate protein-protein interactions, there have been several reports of TCSs that violate this classical model of signal transduction. Our group has recently demonstrated that some of these cross-interacting TCSs function in a manner that imparts a fitness advantage to bacterial pathogens. In this study, we describe interconnectivity between the metabolite-sensing TCSs YpdA/YpdB and BtsS/BtsR in uropathogenic Escherichia coli (UPEC). The YpdA/YpdB and BtsS/BtsR TCSs have been previously reported to interact in K12 E. coli, where they alter the expression of putative transporter genes yhjX and yjiY, respectively. These target genes are both upregulated in UPEC during acute and chronic murine models of urinary tract infection, as well as in response to pyruvate and serine added to growth media in vitro. Here, we show that proper regulation of yhjX in UPEC requires the presence of all components from both of these TCSs. By utilizing plasmid-encoded luciferase reporters tracking the activity of the yhjX and yjiY promoters, we demonstrate that deletions in one TCS substantially alter transcriptional activity of the opposing system's target gene. However, unlike in K12 E. coli, single gene deletions in the YpdA/YpdB system do not alter yjiY gene expression in UPEC, suggesting that niche and lifestyle-specific pressures may be selecting for differential cross-regulation of TCSs in pathogenic and non-pathogenic E. coli.

Adhesive fiber stratification in uropathogenic Escherichia coli biofilms unveils oxygen-mediated control of type 1 pili.

Bacterial biofilms account for a significant number of hospital-acquired infections and complicate treatment options, because bacteria within biofilms are generally more tolerant to antibiotic treatment. This resilience is attributed to transient bacterial subpopulations that arise in response to variations in the microenvironment surrounding the biofilm. Here, we probed the spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli (UPEC), the major causative agent of community-acquired and catheter-associated urinary tract infections. We used matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) to analyze the spatial proteome of intact biofilms in situ. MALDI-TOF IMS revealed protein species exhibiting distinct localizations within surface-associated UPEC biofilms, including two adhesive fibers critical for UPEC biofilm formation and virulence: type 1 pili (Fim) localized exclusively to the air-exposed region, while curli amyloid fibers localized to the air-liquid interface. Comparison of cells grown aerobically, fermentatively, or utilizing an alternative terminal electron acceptor showed that the phase-variable fim promoter switched to the "OFF" orientation under oxygen-deplete conditions, leading to marked reduction of type 1 pili on the bacterial cell surface. Conversely, S pili whose expression is inversely related to fim expression were up-regulated under anoxic conditions. Tethering the fim promoter in the "ON" orientation in anaerobically grown cells only restored type 1 pili production in the presence of an alternative terminal electron acceptor beyond oxygen. Together these data support the presence of at least two regulatory mechanisms controlling fim expression in response to oxygen availability and may contribute to the stratification of extracellular matrix components within the biofilm. MALDI IMS facilitated the discovery of these mechanisms, and we have demonstrated that this technology can be used to interrogate subpopulations within bacterial biofilms.

Biofilm Formation by Uropathogenic Escherichia coli Is Favored under Oxygen Conditions That Mimic the Bladder Environment.

One of the most common urologic problems afflicting millions of people worldwide is urinary tract infection (UTI). The severity of UTIs ranges from asymptomatic bacteriuria to acute cystitis, and in severe cases, pyelonephritis and urosepsis. The primary cause of UTIs is uropathogenic Escherichia coli (UPEC), for which current antibiotic therapies often fail. UPEC forms multicellular communities known as biofilms on urinary catheters, as well as on and within bladder epithelial cells. Biofilm formation protects UPEC from environmental conditions, antimicrobial therapy, and the host immune system. Previous studies have investigated UPEC biofilm formation in aerobic conditions (21% oxygen); however, urine oxygen tension is reduced (4-6%), and urine contains molecules that can be used by UPEC as alternative terminal electron acceptors (ATEAs) for respiration. This study was designed to determine whether these different terminal electron acceptors utilized by E. coli influence biofilm formation. A panel of 50 urine-associated E. coli isolates was tested for the ability to form biofilm under anaerobic conditions and in the presence of ATEAs. Biofilm production was reduced under all tested sub-atmospheric levels of oxygen, with the notable exception of 4% oxygen, the reported concentration of oxygen within the bladder.

The UbiI (VisC) Aerobic Ubiquinone Synthase Is Required for Expression of Type 1 Pili, Biofilm Formation, and Pathogenesis in Uropathogenic Escherichia coli.

UNLABELLED: Uropathogenic Escherichia coli (UPEC), which causes the majority of urinary tract infections (UTI), uses pilus-mediated adherence to initiate biofilm formation in the urinary tract. Oxygen gradients within E. coli biofilms regulate expression and localization of adhesive type 1 pili. A transposon mutant screen for strains defective in biofilm formation identified the ubiI (formerly visC) aerobic ubiquinone synthase gene as critical for UPEC biofilm formation. In this study, we characterized a nonpolar ubiI deletion mutant and compared its behavior to that of wild-type bacteria grown under aerobic and anoxic conditions. Consistent with its function as an aerobic ubiquinone-8 synthase, deletion of ubiI in UPEC resulted in reduced membrane potential, diminished motility, and reduced expression of chaperone-usher pathway pili. Loss of aerobic respiration was previously shown to negatively impact expression of type 1 pili. To determine whether this reduction in type 1 pili was due to an energy deficit, wild-type UPEC and the ubiI mutant were compared for energy-dependent phenotypes under anoxic conditions, in which quinone synthesis is undertaken by anaerobic quinone synthases. Under anoxic conditions, the two strains exhibited wild-type levels of motility but produced diminished numbers of type 1 pili, suggesting that the reduction of type 1 pilus expression in the absence of oxygen is not due to a cellular energy deficit. Acute- and chronic-infection studies in a mouse model of UTI revealed a significant virulence deficit in the ubiI mutant, indicating that UPEC encounters enough oxygen in the bladder to induce aerobic ubiquinone synthesis during infection. IMPORTANCE: The majority of urinary tract infections are caused by uropathogenic E. coli, a bacterium that can respire in the presence and absence of oxygen. The bladder environment is hypoxic, with oxygen concentrations ranging from 4% to 7%, compared to 21% atmospheric oxygen. This work provides evidence that aerobic ubiquinone synthesis must be engaged during bladder infection, indicating that UPEC bacteria sense and use oxygen as a terminal electron acceptor in the bladder and that this ability drives infection potential despite the fact that UPEC is a facultative anaerobe.

Detection and quantification of Babesia species intraerythrocytic parasites by flow cytometry.

OBJECTIVES: Recent work has demonstrated that automated fluorescence flow cytometry (FLC) is a potential alternative for the detection and quantification of Plasmodium parasites. The objective of this study was to apply this novel FLC method to detect and quantify Babesia parasites in venous blood and compare results to light microscopy and polymerase chain reaction methods. METHODS: An automated hematology/malaria analyzer (XN-31; Sysmex) was used to detect and quantify B microti-infected red blood cells from residual venous blood samples (n = 250: Babesia positive, n = 170; Babesia negative, n = 80). As no instrument software currently exists for Babesia, qualitative and quantitative machine learning (ML) algorithms were developed to facilitate analysis. RESULTS: Performance of the ML models was verified against the XN-31 software using P falciparum-infected samples. When applied to Babesia-infected samples, the qualitative ML model demonstrated an area under the curve (AUC) of 0.956 (sensitivity, 95.9%; specificity, 83.3%) relative to polymerase chain reaction. For valid scattergrams, the qualitive model achieved an AUC of 1.0 (sensitivity and specificity, 100%), while the quantitative model demonstrated an AUC of 0.986 (sensitivity, 94.4%; specificity, 100%). CONCLUSIONS: This investigation demonstrates that Babesia parasites can be detected and quantified directly from venous blood using FLC. Although promising, opportunities remain to improve the general applicability of the method.

Immune Adjuvant Therapy With Interleukin-7 in a Lymphopenic Patient With Aplastic Anemia and Mucormycosis.

BACKGROUND: We report the case of a patient with aplastic anemia and pancytopenia on immune-suppressive therapy who developed invasive pulmonary infection with mucormycosis and was treated with immune adjuvant therapy. CASE SUMMARY: Given the patient's profound lymphopenia and progressive invasive mucor despite dual antifungal drug therapy, interleukin (IL)-7, a cytokine that induces lymphocyte activation and proliferation, was instituted and resulted in normalization of absolute lymphocyte counts and was temporally associated with clearance of fungal pathogens and resolution of clinical symptoms. CONCLUSION: Patients with life-threatening fungal infections are frequently immune suppressed and immune adjuvant therapies should be considered in patients who are not responding to antifungal drugs and source control. Well-designed, double-blind, placebo-controlled trials are needed to advance the field. Although a number of immune adjuvants may be beneficial in fungal sepsis, IL-7 is a particularly attractive immune adjuvant because of its broad immunologic effects on key immunologic pathways that mediate enhanced antifungal immune system activity.

Lord of the "rings": A case of Plasmodium falciparum.

Plasmodium falciparum is the most common species to result in severe malaria infection. Examination of blood sample with thick and thin smear is the gold standard diagnostic test and repeat peripheral smear monitoring every 12-24 h should be performed after initiation of treatment to ensure parasite clearance in severe infection. Treatment for severe P. falciparum infection should be initiated without delay, and if artemisinin products are not available, atovaquone-proguanil can be used as an alternative.

Core Genome Multilocus Sequence Typing and Antibiotic Susceptibility Prediction from Whole-Genome Sequence Data of Multidrug-Resistant Pseudomonas aeruginosa Isolates.

Over the past decade, whole-genome sequencing (WGS) has overtaken traditional bacterial typing methods for studies of genetic relatedness. Further, WGS data generated during epidemiologic studies can be used in other clinically relevant bioinformatic applications, such as antibiotic resistance prediction. Using commercially available software tools, the relatedness of 38 clinical isolates of multidrug-resistant Pseudomonas aeruginosa was defined by two core genome multilocus sequence typing (cgMLST) methods, and the WGS data of each isolate was analyzed to predict antibiotic susceptibility to nine antibacterial agents. The WGS typing and resistance prediction data were compared with pulsed-field gel electrophoresis (PFGE) and phenotypic antibiotic susceptibility results, respectively. Simpson's Diversity Index and adjusted Wallace pairwise assessments of the three typing methods showed nearly identical discriminatory power. Antibiotic resistance prediction using a trained analytical pipeline examined 342 bacterial-drug combinations with an overall categorical agreement of 92.4% and very major, major, and minor error rates of 3.6, 4.1, and 4.1%, respectively. IMPORTANCE Multidrug-resistant Pseudomonas aeruginosa isolates are a serious public health concern due to their resistance to nearly all or all of the available antibiotics, including carbapenems. Utilizing molecular approaches in conjunction with antibiotic susceptibility prediction software warrants investigation for use in the clinical laboratory workflow. These molecular tools coupled with antibiotic resistance prediction tools offer the opportunity to overcome the extended turnaround time and technical challenges of phenotypic susceptibility testing.

Understanding and Application of Daptomycin-Susceptible Dose-Dependent Category for Enterococcus: A Mixed-Methods Study.

BACKGROUND: In 2018, the Clinical Microbiology Laboratory at our institution adopted updated daptomycin Enterococcus-susceptible dose-dependent breakpoints. While the introduction of susceptible dose-dependent (SDD) was intended to guide practice toward optimal dosing, the understanding and application of daptomycin SDD breakpoints for enterococci were unknown. METHODS: This mixed-methods study combined a clinician survey with a retrospective pre-post prescribing analysis. An 8-question survey was distributed to infectious diseases (ID) and internal medicine (IM) clinicians. A retrospective chart review of hospitalized adults with infections due to Enterococcus spp. was conducted before (pre-SDD) and after (post-SDD) adoption of SDD reporting for enterococci. RESULTS: Survey response rates were 40 of 98 (41%) for IM and 22 of 34 (65%) for ID clinicians. ID clinicians scored significantly higher than IM clinicians in knowledge of SDD. Chart review of 474 patients (225 pre- vs 249 post-SDD) showed that daptomycin dosage following susceptibility testing was significantly higher post-SDD compared with pre-SDD (8.5 mg/kg vs 6.4 mg/kg; P < .001) with no difference in empiric dosing (6.3 mg/kg vs 6.2 mg/kg; P = .67). Definitive daptomycin use varied between the pre- and post-SDD periods (35.1% vs 16.9%; P < .001). CONCLUSIONS: The survey revealed that ID clinicians placed more importance on and had more confidence in the SDD category over IM clinicians. SDD reporting was associated with a change in definitive daptomycin dosing. ID specialist involvement is recommended in the care of infections due to enterococci for which daptomycin is reported as SDD given their expertise.

Cytochrome bd promotes Escherichia coli biofilm antibiotic tolerance by regulating accumulation of noxious chemicals.

Nutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.

Performance of three polymerase chain reaction-based assays for detection of SARS-CoV-2 in different upper respiratory tract specimens.

To meet the testing demands and overcome supply chain issues during the SARS-CoV-2 pandemic, many clinical laboratories validated multiple SARS-CoV-2 molecular testing platforms. Here, we compare three different molecular assays for SARS-CoV-2 that received emergency use authorization (EUA) from the U.S. Food and Drug Administration. In order to determine the agreement among Roche cobas® SARS-CoV-2 Test (Cobas), Abbott RealTime SARS-CoV-2 assay (ART), and Mayo Clinic Laboratory SARS-CoV-2 Molecular Detection Assay (Mayo LDT), 100 each of anterior nares (AN), nasopharyngeal (NP), oropharyngeal (OP), and NP+OP swabs were tested on each platform. The consensus result was defined as agreement by 2 or more methods. Furthermore, 30 positive NP swabs from each molecular platform (n = 90 total) were tested on the three platforms to determine the PPA among positive samples. ART platform called more specimens positive than the other two platforms. All three assays performed with greater than 90% agreement for NP specimens throughout the study.

Initial SARS-CoV-2 PCR crossing point does not predict hospitalization and duration of PCR positivity.

This study aimed to determine if the crossing point of the initial positive SARS-CoV-2 PCR test correlated with patient demographics, subsequent hospitalization, or duration of positivity. Seventy-three patients with two or more positive PCR tests had a median time of 23 days to two consecutive negative results.

Data highlighting phenotypic diversity of urine-associated Escherichia coli isolates.

This article provides a reusable dataset describing detailed phenotypic and associated clinical parameters in n=303 clinical isolates of urinary Escherichia coli collected at Vanderbilt University Medical Center. De-identified clinical data collected with each isolate are detailed here and correlated to biofilm abundance and metabolomics data. Biofilm-abundance data were collected for each isolate under different in vitro conditions along with datasets quantifying biofilm abundance of each isolate under different conditions. Metabolomics data were collected from a subset of bacterial strains isolated from uncomplicated cases of cystitis or cases with no apparent symptoms accompanying colonization. For more insight, please see "Defining a Molecular Signature for Uropathogenic versus Urocolonizing Escherichia coli: The Status of the Field and New Clinical Opportunities" [1].

Defining a Molecular Signature for Uropathogenic versus Urocolonizing Escherichia coli: The Status of the Field and New Clinical Opportunities.

Urinary tract infections (UTIs) represent a major burden across the population, although key facets of their pathophysiology and host interaction remain unclear. Escherichia coli epitomizes these obstacles: this gram-negative bacterial species is the most prevalent agent of UTIs worldwide and can also colonize the urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB). Unfortunately, at the level of the individual E. coli strains, the relationship between UTI and ASB is poorly defined, confounding our understanding of microbial pathogenesis and strategies for clinical management. Unlike diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli (UPEC) remains phenomenologic, without conserved phenotypes and known genetic determinants that rigorously distinguish UTI- and ASB-associated strains. This article provides a cross-disciplinary review of the current issues from interrelated mechanistic and diagnostic perspectives and describes new opportunities by which clinical resources can be leveraged to overcome molecular challenges. Specifically, we present our work harnessing a large collection of patient-derived isolates to identify features that do (and do not) distinguish UTI- from ASB-associated E. coli strains. Analyses of biofilm formation, previously reported to be higher in ASB strains, revealed extensive phenotypic heterogeneity that did not correlate with symptomatology. However, metabolomic experiments revealed distinct signatures between ASB and cystitis isolates, including in the purine pathway (previously shown to be critical for intracellular survival during acute infection). Together, these studies demonstrate how large-scale, wild-type approaches can help dissect the physiology of colonization versus infection, suggesting that the molecular definition of UPEC may rest at the level of global bacterial metabolism.