Inpatient clinicians' approach to diagnosis of urinary tract infections in older adults using the COM-B model: a qualitative assessment.

Our interviews of inpatient clinicians (physicians, physician assistants) modeled after the Capability, Opportunity, and Motivation Model of Behavior model revealed opportunity and motivation as important drivers for overdiagnosis and overprescribing for asymptomatic bacteriuria in older adults. Understanding these barriers is an important step toward implementing age-friendly stewardship interventions.

Presence of vitamin B12 metabolism in the last common ancestor of land plants.

Vitamin B12, also known as cobalamin, is an essential organic cofactor for methionine synthase (METH), and is only synthesized by a subset of bacteria. Plants and fungi have an alternative methionine synthase (METE) that does not need B12 and are typically considered not to utilize it. Some algae facultatively utilize B12 because they encode both METE and METH, while other algae are dependent on B12 as they encode METH only. We performed phylogenomic analyses of METE, METH and 11 further proteins involved in B12 metabolism across more than 1600 plant and algal genomes and transcriptomes (e.g. from OneKp), demonstrating the presence of B12-associated metabolism deep into the streptophytes. METH and five further accessory proteins (MTRR, CblB, CblC, CblD and CblJ) were detected in the hornworts (Anthocerotophyta), and two (CblB and CblJ) were identified in liverworts (Marchantiophyta) in the bryophytes, suggesting a retention of B12-metabolism in the last common land plant ancestor. Our data further show more limited distributions for other B12-related proteins (MCM and RNR-II) and B12 dependency in several algal orders. Finally, considering the collection sites of algae that have lost B12 metabolism, we propose freshwater-to-land transitions and symbiotic associations to have been constraining factors for B12 availability in early plant evolution. This article is part of the theme issue 'The evolution of plant metabolism'.

Evidence gaps among systematic reviews examining the relationship of race, ethnicity, and social determinants of health with adult inpatient quality measures.

Background: The field of healthcare epidemiology is increasingly focused on identifying, characterizing, and addressing social determinants of health (SDOH) to address inequities in healthcare quality. To identify evidence gaps, we examined recent systematic reviews examining the association of race, ethnicity, and SDOH with inpatient quality measures. Methods: We searched Medline via OVID for English language systematic reviews from 2010 to 2022 addressing race, ethnicity, or SDOH domains and inpatient quality measures in adults using specific topic questions. We imported all citations to Covidence (www.covidence.org, Veritas Health Innovation) and removed duplicates. Two blinded reviewers assessed all articles for inclusion in 2 phases: title/abstract, then full-text review. Discrepancies were resolved by a third reviewer. Results: Of 472 systematic reviews identified, 39 were included. Of these, 23 examined all-cause mortality; 6 examined 30-day readmission rates; 4 examined length of stay, 4 examined falls, 2 examined surgical site infections (SSIs) and one review examined risk of venous thromboembolism. The most evaluated SDOH measures were sex (n = 9), income and/or employment status (n = 9), age (n = 6), race and ethnicity (n = 6), and education (n = 5). No systematic reviews assessed medication use errors or healthcare-associated infections. We found very limited assessment of other SDOH measures such as economic stability, neighborhood, and health system access. Conclusion: A limited number of systematic reviews have examined the association of race, ethnicity and SDOH measures with inpatient quality measures, and existing reviews highlight wide variability in reporting. Future systematic evaluations of SDOH measures are needed to better understand the relationships with inpatient quality measures.

Time to Positive Blood Cultures Among Critically Ill Children Admitted to the PICU.

OBJECTIVES: Our study aimed to assess the time to positivity (TTP) of clinically significant blood cultures in critically ill children admitted to the PICU. DESIGN: Retrospective review of positive blood cultures in patients admitted or transferred to the PICU. SETTING: Large tertiary-care medical center with over 90 PICU beds. PATIENTS: Patients 0-20 years old with bacteremia admitted or transferred to the PICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was the TTP, defined as time from blood culture draw to initial Gram stain result. Secondary endpoints included percentage of cultures reported by elapsed time, as well as the impact of pathogen and host immune status on TTP. Host immune status was classified as previously healthy, standard risk, or immunocompromised. Linear regression for TTP was performed to account for age, blood volume, and Gram stain. Among 164 episodes of clinically significant bacteremia, the median TTP was 13.3 hours (interquartile range, 10.7-16.8 hr). Enterobacterales, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus pneumoniae were most commonly identified. By 12, 24, 36, and 48 hours, 37%, 89%, 95%, and 97% of positive cultures had resulted positive, respectively. Median TTP stratified by host immune status was 13.2 hours for previously healthy patients, 14.0 hours for those considered standard risk, and 10.6 hours for immunocompromised patients (p = 0.001). Median TTP was found to be independent of blood volume. No difference was seen in TTP for Gram-negative vs. Gram-positive organisms (12.2 vs. 13.9 hr; p = 0.2). CONCLUSIONS: Among critically ill children, 95% of clinically significant blood cultures had an initial positive result within 36 hours, regardless of host immune status. Need for antimicrobial therapy should be frequently reassessed and implementation of a shorter duration of empiric antibiotics should be considered in patients with low suspicion for infection.

Beyond Infection: Mortality and End-of-Life Care Associated With Infectious Disease Consultation in an Academic Health System.

BACKGROUND: Infectious diseases (ID) physicians are increasingly faced with the challenge of caring for patients with terminal illnesses or incurable infections. METHODS: This was a retrospective cohort of all patients with an ID consult within an academic health system from 1 January 2014 through 31 December 2023, including community, general, and transplant ID consult services. RESULTS: There were 60 820 inpatient ID consults (17 235 community, 29 999 general, and 13 586 transplant) involving 37 848 unique patients. The number of consults increased by 94% and the rate rose from 5.0 to 9.9 consults per 100 inpatients (P < .001). In total, 7.5% of patients receiving an ID consult died during admission and 1006 (2.6%) of patients were discharged to hospice. In-hospital mortality was 5.2% for community ID, 7.8% for general ID, and 10.7% for transplant ID patients (P < .001). Six-month mortality was 9% for all nonobstetric admissions versus 19% for community ID, 20.9% for general ID, and 22.3% for transplant ID. In total 2866 (7.6%) of all patients receiving ID consultation also received palliative care consultation during the same hospitalization. The index ID consult preceded any palliative consult in the majority (69.5%) of cases. A total of 16.3% of patients had a do-not-resuscitate order during the index hospitalization; 12.2% of all patients with a do-not-resuscitate order had this placed on the same day as the ID consult. CONCLUSIONS: Patients receiving ID consultation were increasingly complex and more likely to die soon after consultation. These results provide a framework for ID clinicians to consider their role in end-of-life care.

Clinical outcomes in patients with piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae bloodstream infection.

BACKGROUND: A small proportion of Escherichia coli and Klebsiella pneumoniae demonstrate in vitro non-susceptibility to piperacillin/tazobactam but retain susceptibility to ceftriaxone. Uncertainty remains regarding how best to treat these isolates. OBJECTIVES: We sought to compare clinical outcomes between patients with piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae bloodstream infection receiving definitive therapy with ceftriaxone versus an alternative effective antibiotic. METHODS: We retrospectively identified patients with a positive blood culture for piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae between 1 January 2013 and 31 December 2022. Patients were divided into one of two definitive treatment groups: ceftriaxone or alternative effective antibiotic. Our primary outcome was a composite of 90 day all-cause mortality, hospital readmission, or recurrence of infection. We used Cox proportional hazards models to compare time with the composite outcome between groups. RESULTS: Sixty-two patients were included in our analysis. Overall, median age was 63 years (IQR 49.5-71.0), the most common source of infection was intra-abdominal (25/62; 40.3%) and the median total duration of therapy was 12.0 days (IQR 9.0-16.8). A total of 9/22 (40.9%) patients in the ceftriaxone treatment group and 18/40 (45.0%) patients in the alternative effective antibiotic group met the composite endpoint. In an adjusted time-to-event analysis, there was no difference in the composite endpoint between groups (HR 0.67, 95% CI 0.30-1.50). The adjusted Bayesian posterior probability that the HR was less than or equal to 1 (i.e. ceftriaxone is as good or better than alternative therapy) was 85%. CONCLUSIONS: These findings suggest that ceftriaxone can be used to effectively treat bloodstream infections with E. coli or K. pneumoniae that are non-susceptible to piperacillin/tazobactam but susceptible to ceftriaxone.

Light management by algal aggregates in living photosynthetic hydrogels.

Rapid progress in algal biotechnology has triggered a growing interest in hydrogel-encapsulated microalgal cultivation, especially for the engineering of functional photosynthetic materials and biomass production. An overlooked characteristic of gel-encapsulated cultures is the emergence of cell aggregates, which are the result of the mechanical confinement of the cells. Such aggregates have a dramatic effect on the light management of gel-encapsulated photobioreactors and hence strongly affect the photosynthetic outcome. To evaluate such an effect, we experimentally studied the optical response of hydrogels containing algal aggregates and developed optical simulations to study the resultant light intensity profiles. The simulations are validated experimentally via transmittance measurements using an integrating sphere and aggregate volume analysis with confocal microscopy. Specifically, the heterogeneous distribution of cell aggregates in a hydrogel matrix can increase light penetration while alleviating photoinhibition more effectively than in a flat biofilm. Finally, we demonstrate that light harvesting efficiency can be further enhanced with the introduction of scattering particles within the hydrogel matrix, leading to a fourfold increase in biomass growth. Our study, therefore, highlights a strategy for the design of spatially efficient photosynthetic living materials that have important implications for the engineering of future algal cultivation systems.

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

Combined cerebrospinal fluid metabolomic and cytokine profiling in tuberculosis meningitis reveals robust and prolonged changes in immunometabolic networks.

Much of the high mortality in tuberculosis meningitis (TBM) is attributable to excessive inflammation, making it imperative to identify targets for host-directed therapies that reduce pathologic inflammation and mortality. In this study, we investigate how cytokines and metabolites in the cerebral spinal fluid (CSF) associate with TBM at diagnosis and during TBM treatment. At diagnosis, TBM patients (n = 17) demonstrate significant increases of cytokines and chemokines that promote inflammation and cell migration including IL-17A, IL-2, TNFα, IFNγ, and IL-1ß versus asymptomatic controls without known central nervous system pathology (n = 20). Inflammatory immune signaling had a strong positive correlation with immunomodulatory metabolites including kynurenine, lactic acid, and carnitine and strong negative correlations with tryptophan and itaconate. Inflammatory immunometabolic networks were only partially reversed with two months of effective TBM treatment and remained significantly different compared to CSF from controls. Together, these data highlight a critical role for host metabolism in regulating the inflammatory response to TBM and indicate the timeline for restoration of immune homeostasis in the CSF is prolonged.

Overexpression of PSR1 in Chlamydomonas reinhardtii induces luxury phosphorus uptake.

Remediation using micro-algae offers an attractive solution to environmental phosphate (PO4 3-) pollution. However, for maximum efficiency, pre-conditioning of algae to induce 'luxury phosphorus (P) uptake' is needed. To replicate this process, we targeted the global regulator PSR1 (Myb transcription factor: Phosphate Starvation Response 1) for over-expression in algae. Manipulating a single gene (PSR1) drove uptake of both PO4 3- and a Mg2+ counter-ion leading to increased PolyP granule size, raising P levels 4-fold to 8% dry cell weight, and accelerated removal of PO4 3- from the medium. Examination of the gene expression profile showed that the P-starvation response was mimicked under P-replete conditions, switching on luxury uptake. Hyper-accumulation of P depended on a feed-forward mechanism, where a small set of 'Class I' P-transporter genes were activated despite abundant external PO4 3- levels. The transporters drove a reduction in external PO4 3- levels, permitting more genes to be expressed (Class II), leading to more P-uptake. Our data pointed toward a PSR1-independent mechanism for detection of external PO4 3- which suppressed Class II genes. This model provided a plausible mechanism for P-overplus where prior P-starvation elevates PSR1 and on P-resupply causes luxury P-uptake. This is because the Class I genes, which include P-transporter genes, are not suppressed by the excess PO4 3-. Taken together, these discoveries facilitate a bio-circular approach of recycling nutrients from wastewater back to agriculture.

Combined cerebrospinal fluid metabolomic and cytokine profiling in tuberculosis meningitis reveals robust and prolonged changes in immunometabolic networks.

Much of the high mortality in tuberculosis meningitis (TBM) is attributable to excessive inflammation, making it imperative to identify targets for host-directed therapies that reduce pathologic inflammation and mortality. In this study, we investigate how cytokines and metabolites in the cerebral spinal fluid (CSF) associate with TBM at diagnosis and during TBM treatment. At diagnosis, TBM patients demonstrate significant increases versus controls of cytokines and chemokines that promote inflammation and cell migration including IL-17A, IL-2, TNFα, IFNγ, and IL-1ß. Inflammatory immune signaling was strongly correlated with immunomodulatory metabolites including kynurenine, lactic acid, carnitine, tryptophan, and itaconate. Inflammatory immunometabolic networks were only partially reversed with two months of effective TBM treatment and remained significantly different versus control CSF. Together, these data highlight a critical role for host metabolism in regulating the inflammatory response to TBM and indicate the timeline for restoration of immune homeostasis in the CSF is prolonged.

Microfluidic preparation of composite hydrogel microparticles for the staining of microalgal cells.

The delivery of lipophilic dyes, such as BODIPY 505/515, to cells is often hindered by their low aqueous solubility, necessitating the use of organic solvents to facilitate the delivery, which unfortunately compromises the viability of the cells. In this work, we demonstrate the generation of novel composite hydrogel microparticles loaded with BODIPY 505/515, which can be used to deliver the dye to microalgal cells to stain the intracellular lipids. The microparticles were prepared by combining polymeric micelles with hydrogel technology to obtain microparticles of enhanced loading capacity. The generated hydrogel microparticles were tested by incubation with Phaeodactylum tricornutum algal cells. The cells were rapidly and successfully stained by the dye-containing microparticles, and their viability was not affected by the staining process. The method can also be used to stain other types of microalgal cells, such as Nannochloropsis gaditana cells. We therefore believe that this work offers a versatile and useful solution to important cell-staining problems in biotechnology.

Cerebrospinal fluid concentrations of fluoroquinolones and carbapenems in tuberculosis meningitis.

Background: Tuberculosis meningitis (TBM) is the most lethal form of TB. It is difficult to treat in part due to poor or uncertain drug penetration into the central nervous system (CNS). To help fill this knowledge gap, we evaluated the cerebrospinal fluid (CSF) concentrations of fluoroquinolones and carbapenems in patients being treated for TBM. Methods: Serial serum and CSF samples were collected from hospitalized patients being treated for TBM. CSF was collected from routine lumbar punctures between alternating timepoints of 2 and 6 h after drug administration to capture early and late CSF penetration. Rich serum sampling was collected after drug administration on day 28 for non-compartmental analysis. Results: Among 22 patients treated for TBM (8 with confirmed disease), there was high use of fluoroquinolones (levofloxacin, 21; moxifloxacin, 10; ofloxacin, 6) and carbapenems (imipenem, 11; meropenem, 6). Median CSF total concentrations of levofloxacin at 2 and 6 h were 1.34 mg/L and 3.36 mg/L with adjusted CSF/serum ratios of 0.41 and 0.63, respectively. For moxifloxacin, the median CSF total concentrations at 2 and 6 h were 0.78 mg/L and 1.02 mg/L with adjusted CSF/serum ratios of 0.44 and 0.62. Serum and CSF concentrations of moxifloxacin were not affected by rifampin use. Among the 76 CSF samples measured for carbapenem concentrations, 79% were undetectable or below the limit of detection. Conclusion: Fluoroquinolones demonstrated high CSF penetration indicating their potential usefulness for the treatment of TBM. Carbapenems had lower than expected CSF concentrations.

The Impact of Diabetes and Prediabetes on Prevalence of Mycobacterium tuberculosis Infection Among Household Contacts of Active Tuberculosis Cases in Ethiopia.

Background: It is uncertain whether diabetes affects the risk of developing latent tuberculosis infection (LTBI) following exposure to Mycobacterium tuberculosis (Mtb). We assessed the relationship of diabetes or prediabetes and LTBI among close and household contacts (HHCs) of patients with active pulmonary tuberculosis (TB) disease in Addis Ababa, Ethiopia. Methods: In this cross-sectional study, we performed interferon-γ release assays, TB symptom screening, and point-of-care glycolated hemoglobin (HbA1c) testing among HHCs of active TB cases. Diabetes status was classified into diabetes (HbA1c ≥6.5% or self-reported diagnosis), prediabetes (5.7%-6.4%), and euglycemia (≤5.6%). Multivariable logistic regression was used to determine the association of diabetes with LTBI. Results: Among 597 study participants, 123 (21%) had dysglycemia including diabetes (n = 31) or prediabetes (n = 92); 423 (71%) participants were diagnosed with LTBI. Twelve of 31 (39%) HHCs with diabetes were previously undiagnosed with diabetes. The prevalence of LTBI among HHCs with diabetes, prediabetes, and euglycemia was 87% (27/31), 73% (67/92), and 69% (329/474), respectively. In multivariable analysis adjusted for age, sex, and HIV status, the odds of LTBI among HHCs with diabetes were 2.33 (95% confidence interval [CI], .76-7.08) times the odds of LTBI without diabetes. When assessing interaction with age, the association of diabetes and LTBI was robust among participants aged ≥40 years (adjusted odds ratio [aOR], 3.68 [95% CI, .77-17.6]) but not those <40 years (aOR, 1.15 [95% CI, .22-6.1]). Conclusions: HHCs with diabetes may be more likely to have LTBI than those with euglycemia. Further investigations are needed to assess mechanisms by which diabetes may increase risk of LTBI after Mtb exposure.

Long term outcomes of patients with tuberculous meningitis: The impact of drug resistance.

BACKGROUND: Little is known about the impact of drug-resistance on clinical outcomes among patients with tuberculosis meningitis (TBM). METHODS: A retrospective cohort study among patients treated for TBM in Tbilisi, Georgia. We performed medical chart abstraction to collect patient data. Long-term vital status was assessed using the Georgia National Death Registry. We utilized a Cox proportional-hazards model to evaluate the association of drug-resistance and mortality. RESULTS: Among 343 TBM suspects, 237 had a presentation consistent with TBM. Drug resistance was suspected (n = 5) or confirmed (n = 31) in 36 patients including 30 with multidrug- or rifampin-resistance and 6 with isoniazid-resistance. Thirty-four patients had HIV. The median follow-up time was 1331 days (IQR, 852-1767). Overall, 73 of 237 (30%) people died with 50 deaths occurring during and 23 after treatment. The proportion of death was higher among patients with drug-resistant vs. drug-susceptible disease (67% vs. 24%, p<0.001) and with HIV versus no HIV (59% vs 27%, p<0.001). Mortality was significantly higher in patients with drug-resistant TBM after 90 days of treatment (aHR = 7.2, CI95% [3.6-14.3], p < 0.001). CONCLUSIONS: Mortality was high among patients with drug-resistant TBM with many deaths occurring post treatment. More effective treatment options are urgently needed for drug-resistant TBM.

Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches.

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little information is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP-P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Low rates of antibiotic use among ambulatory patients with coronavirus disease 2019 (COVID-19).

We assessed the prevalence of antibiotic prescriptions among ambulatory patients tested for coronavirus disease 2019 (COVID-19) in a large public US healthcare system and found a low overall rate of antibiotic prescriptions (6.7%). Only 3.8% of positive severe acute respiratory coronavirus virus 2 (SARS-CoV-2) tests were associated with an antibiotic prescription within 7 days.

Exploring the onset of B12 -based mutualisms using a recently evolved Chlamydomonas auxotroph and B12 -producing bacteria.

Cobalamin (vitamin B12 ) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B12 . Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12 -dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12 -independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12 -dependent algae.

CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome.

The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( = plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations. This presents issues with respect to selectable markers, since there are a limited number available, they can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative selection system, known as CpPosNeg, for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (AadA: conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (CodA: conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the 3'UTR of the marker and the 3'UTR of the introduced transgene. We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.

Cycloserine and Linezolid for Tuberculosis Meningitis: Pharmacokinetic Evidence of Potential Usefulness.

BACKGROUND: The ability of antituberculosis drugs to cross the blood-brain barrier and reach the central nervous system is critical to their effectiveness in treating tuberculosis meningitis (TBM). We sought to fill a critical knowledge gap by providing data on the ability of new and repurposed antituberculosis drugs to penetrate into the cerebrospinal fluid (CSF). METHODS: We conducted a clinical pharmacology study among patients treated for TBM in Tbilisi, Georgia, from January 2019 until January 2020. Serial serum and CSF samples were collected while patients were hospitalized. CSF was collected from routine lumbar punctures with the timing of the lumbar puncture alternating between 2 and 6 hours to capture early and late CSF penetration. RESULTS: A total of 17 patients treated for TBM (8 with confirmed disease) were included; all received linezolid, with a subset receiving cycloserine (5), clofazimine (5), delamanid (4), and bedaquiline (2). All CSF measurements of bedaquiline (12), clofazimine (24), and delamanid (19) were below the limit of detection. The median CSF concentrations of cycloserine at 2 and 6 hours were 15.90 and 15.10 µg/mL with adjusted CSF/serum ratios of 0.52 and 0.66. CSF concentrations of linezolid were 0.90 and 3.14 µg/mL at 2 and 6 hours, with adjusted CSF/serum ratios of 0.25 and 0.59, respectively. CSF serum linezolid concentrations were not affected by rifampin coadministration. CONCLUSIONS: Based on moderate to high CSF penetration, linezolid and cycloserine may be effective drugs for TBM treatment, whereas the utility of bedaquiline, delamanid, and clofazimine is uncertain given their low CSF penetration.