Whole-genome sequence of Mucor velutinosus NIH1002, a strain associated with disseminated disease.

The draft genome of Mucor velutinosus NIH1002, a 2011 isolate from a case of disseminated disease, was sequenced using PacBio long-read and HiSeq short-read technologies. The genome has 43 contigs, an N50 of 2.65 Mb, and 13,295 protein-coding genes. It is the most complete M. velutinosus genome to date.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Within-Host Evolution of Bacterial Pathogens in Acute and Chronic Infection.

Bacterial pathogens undergo remarkable adaptive change in response to the selective forces they encounter during host colonization and infection. Studies performed over the past few decades have demonstrated that many general evolutionary processes can be discerned during the course of host adaptation, including genetic diversification of lineages, clonal succession events, convergent evolution, and balanced fitness trade-offs. In some cases, elevated mutation rates resulting from mismatch repair or proofreading deficiencies accelerate evolution, and active mobile genetic elements or phages may facilitate genome plasticity. The host immune response provides another critical component of the fitness landscapes guiding adaptation, and selection operating on pathogens at this level may lead to immune evasion and the establishment of chronic infection. This review summarizes recent advances in this field, with a special focus on different forms of bacterial genome plasticity in the context of infection, and considers clinical consequences of adaptive changes for the host.

The Host Adapted Fungal Pathogens of Pneumocystis Genus Utilize Genic Regional Centromeres.

Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of Schizosaccharomyces pombe. Using organisms from a short-term in vitro culture or infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.

Roving methyltransferases generate a mosaic epigenetic landscape and influence evolution in Bacteroides fragilis group.

Three types of DNA methyl modifications have been detected in bacterial genomes, and mechanistic studies have demonstrated roles for DNA methylation in physiological functions ranging from phage defense to transcriptional control of virulence and host-pathogen interactions. Despite the ubiquity of methyltransferases and the immense variety of possible methylation patterns, epigenomic diversity remains unexplored for most bacterial species. Members of the Bacteroides fragilis group (BFG) reside in the human gastrointestinal tract as key players in symbiotic communities but also can establish anaerobic infections that are increasingly multi-drug resistant. In this work, we utilize long-read sequencing technologies to perform pangenomic (n = 383) and panepigenomic (n = 268) analysis of clinical BFG isolates cultured from infections seen at the NIH Clinical Center over four decades. Our analysis reveals that single BFG species harbor hundreds of DNA methylation motifs, with most individual motif combinations occurring uniquely in single isolates, implying immense unsampled methylation diversity within BFG epigenomes. Mining of BFG genomes identified more than 6000 methyltransferase genes, approximately 1000 of which were associated with intact prophages. Network analysis revealed substantial gene flow among disparate phage genomes, implying a role for genetic exchange between BFG phages as one of the ultimate sources driving BFG epigenome diversity.

Association between minimum inhibitory concentration values and mortality risk in patients with Stenotrophomonas maltophilia infections: a retrospective cohort study of electronic health records from 148 US hospitals.

Background: Clinical data informing antimicrobial susceptibility breakpoints for Stenotrophomonas maltophilia infections are lacking. We sought to leverage real-world data to identify MIC values within the currently defined susceptible range that could discriminate mortality risk for patients with S. maltophilia infections and guide future breakpoint revisions. Methods: Inpatients with S. maltophilia infection who received single-agent targeted therapy with levofloxacin or trimethoprim/sulfamethoxazole were identified in the Cerner HealthFacts electronic health record database. Encounters were restricted to those with MIC values reported to be in the susceptible range for both agents. Curation for exact (non-range) MIC values yielded sequentially granular model populations. Logistic regression was used to calculate adjusted OR (aOR) of mortality or hospice discharge associated with different susceptible-range MICs, controlling for patient- and centre-related factors, and infection site, polymicrobial infection and receipt of empirical therapy. Results: Seventy-three of 851 levofloxacin-treated patients had levofloxacin MIC of exactly 2 mg/L (current Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoint) and served as the reference category for levofloxacin breakpoint models. In breakpoint model I (n = 501), aOR of mortality associated with infection due to isolates with levofloxacin MIC of ≤1 versus 2 mg/L were similar [aOR = 1.79 (95% CI 0.88-3.62), P = 0.11]. In breakpoint model IIa (n = 358), aOR of mortality associated with MIC ≤0.5 versus 2 mg/L were also similar [aOR 0.1.36 (95% CI 0.65-2.83), P = 0.41]. However, breakpoint model IIb (n = 297) displayed higher aOR of mortality associated with an MIC of 1 versus 2 mg/L [aOR 2.36 (95% CI 1.14-4.88), P = 0.02]. Only 9/645 trimethoprim/sulfamethoxazole-treated patients had trimethoprim/sulfamethoxazole MIC of exactly 2/38 mg/L precluding informative models for this agent. Conclusions: In this retrospective study of real-world patients with S. maltophilia infection, risk-adjusted survival data do not appear to stratify patients clinically within current susceptible-range MIC breakpoint for levofloxacin (≤2 mg/L) by mortality.

Association between piperacillin/tazobactam MIC and survival among hospitalized patients with Enterobacterales infections: retrospective cohort analysis of electronic health records from 161 US hospitals.

Introduction: A recent randomized trial has suggested an increased risk of mortality for ceftriaxone-non-susceptible Enterobacterales infections treated with piperacillin/tazobactam compared with meropenem despite MICs within the susceptible range. Methods: We conducted a retrospective cohort study of clinical encounters within the Cerner Health Facts database to identify all encounters between 2001 and 2017 in which Enterobacterales infections were treated empirically with piperacillin/tazobactam and for which MICs to the drug were available. Multivariate regression analysis was performed to enable partitioning of MICs into discrete strata based on statistically significant difference in mortality risk. Results: During the study period, 10 101 inpatient encounters were identified meeting inclusion criteria. The crude in-hospital mortality for the entire cohort was 16.5%. Partitioning analysis identified a breakpoint of ≤16/4 mg/L that dichotomized encounters into lower versus higher mortality risk strata in the primary cohort of overall infections. This finding persisted in sequentially granular subsets where specific MICs ≤8/4 mg/L were reported (in lieu of ranges) as well as in the high-reliability subset with bloodstream infections. A higher clinical breakpoint of ≥128/4 mg/L dichotomized encounters with respiratory tract infection. No breakpoint was identified when restricting to encounters with urinary tract infections, ICU admits or upon restricting analysis to encounters with ceftriaxone-resistant isolates. Conclusions: Clinical data suggest improved outcomes when piperacillin/tazobactam is prescribed for Enterobacterales infections with an MIC of ≤16/4 mg/L compared with ≥32/4 mg/L.

Comparative Evaluation of Current Biochemical-, Sequencing-, and Proteomic-Based Identification Methods for the Streptococcus bovis Group.

The Streptococcus bovis group (previously group D streptococci) consists of seven distinct species and subspecies. Definitive identification within the group is important, as certain organisms have been associated with gastrointestinal carcinoma, bacteremia, infective endocarditis, meningitis, biliary tract disease, and carcinoma, among others. Definitive identification, however, remains elusive due to limitations and inconsistencies across commonly used identification platforms in the United States. Here, we compared the performance of standard biochemical (Trek Gram-positive identification [GPID] plate, Vitek 2 GPID), sequencing (16S rDNA, sodA) databases (NCBI, RDP, CDC MicrobeNet), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) platforms (Vitek MS, Bruker Biotyper MS) using a set of eight type strains representing all seven strains within the S. bovis group. Despite the evaluation of contemporary methods, no single platform was able to definitively identify all type strains within the S. bovis group. Vitek MS (85.7%, 7/8) provided the most accurate definitive identifications, followed by sodA sequencing (75%, 6/8). Vitek 2 and Bruker Biotyper RUO platforms performed the next best (62.5%, 5/8). All remaining platforms failed to adequately differentiate type strains within the S. bovis group (range, 0 to 37.5%). Laboratorians and clinicians should be aware of the identification limitations of routine testing algorithms and incorporate reflex testing, when appropriate, to platforms such as Vitek MS and/or sodA sequencing that are more able to definitively identify S. bovis group organisms. Further clinical evaluation was conducted using 65 clinical isolates from three geographically distinct U.S. institutions. Future improvements in identification platforms may reveal new clinical and epidemiological trends for members of the S. bovis group.

Hypermutator strains of Pseudomonas aeruginosa reveal novel pathways of resistance to combinations of cephalosporin antibiotics and beta-lactamase inhibitors.

Hypermutation due to DNA mismatch repair (MMR) deficiencies can accelerate the development of antibiotic resistance in Pseudomonas aeruginosa. Whether hypermutators generate resistance through predominantly similar molecular mechanisms to wild-type (WT) strains is not fully understood. Here, we show that MMR-deficient P. aeruginosa can evolve resistance to important broad-spectrum cephalosporin/beta-lactamase inhibitor combination antibiotics through novel mechanisms not commonly observed in WT lineages. Using whole-genome sequencing (WGS) and transcriptional profiling of isolates that underwent in vitro adaptation to ceftazidime/avibactam (CZA), we characterized the detailed sequence of mutational and transcriptional changes underlying the development of resistance. Surprisingly, MMR-deficient lineages rapidly developed high-level resistance (>256 µg/mL) largely without corresponding fixed mutations or transcriptional changes in well-established resistance genes. Further investigation revealed that these isolates had paradoxically generated an early inactivating mutation in the mexB gene of the MexAB-OprM efflux pump, a primary mediator of CZA resistance in P. aeruginosa, potentially driving an evolutionary search for alternative resistance mechanisms. In addition to alterations in a number of genes not known to be associated with resistance, 2 mutations were observed in the operon encoding the RND efflux pump MexVW. These mutations resulted in a 4- to 6-fold increase in resistance to ceftazidime, CZA, cefepime, and ceftolozane-tazobactam when engineered into a WT strain, demonstrating a potentially important and previously unappreciated mechanism of resistance to these antibiotics in P. aeruginosa. Our results suggest that MMR-deficient isolates may rapidly evolve novel resistance mechanisms, sometimes with complex dynamics that reflect gene inactivation that occurs with hypermutation. The apparent ease with which hypermutators may switch to alternative resistance mechanisms for which antibiotics have not been developed may carry important clinical implications.

Disseminated Mycoplasma Orale Infection in Patients With Phosphoinositide-3-Kinase Regulatory Subunit 1 Mutations.

Mycoplasma orale is a rare cause of invasive infection in immunodeficient hosts. Phosphatidylinositol 3-kinase, regulatory subunit 1 (PI3KR1) mutations predispose patients to sinopulmonary infections, alongside bronchiectasis autoimmunity and lymphoproliferation. We report 2 cases of PI3KR1 deficiency with invasive M orale and effective treatment options.

Trimethoprim-Sulfamethoxazole Versus Levofloxacin for Stenotrophomonas maltophilia Infections: A Retrospective Comparative Effectiveness Study of Electronic Health Records from 154 US Hospitals.

BACKGROUND: Trimethoprim-sulfamethoxazole (TMP-SMX) is considered first-line therapy for Stenotrophomonas maltophilia infections based on observational data from small studies. Levofloxacin has emerged as a popular alternative due to tolerability concerns related to TMP-SMX. Data comparing levofloxacin to TMP-SMX as targeted therapy are lacking. METHODS: Adult inpatient encounters January 2005 through December 2017 with growth of S maltophilia in blood and/or lower respiratory cultures were identified in the Cerner Healthfacts database. Patients included received targeted therapy with either levofloxacin or TMP-SMX. Overlap weighting was used followed by downstream weighted regression. The primary outcome was adjusted odds ratio (aOR) for in-hospital mortality or discharge to hospice. The secondary outcome was number of days from index S maltophilia culture to hospital discharge. RESULTS: Among 1581 patients with S maltophilia infections, levofloxacin (n = 823) displayed statistically similar mortality risk (aOR, 0.76 [95% confidence interval {CI}, .58-1.01]; P = .06) compared to TMP-SMX (n = 758). Levofloxacin (vs TMP-SMX) use was associated with a lower aOR of death in patients with lower respiratory tract infection (n = 1452) (aOR, 0.73 [95% CI, .54-.98]; P = .03) and if initiated empirically (n = 89) (aOR, 0.16 [95% CI, .03-.95]; P = .04). The levofloxacin cohort had fewer hospital days between index culture collection and discharge (weighted median [interquartile range], 7 [4-13] vs 9 [6-16] days; P < .0001). CONCLUSIONS: Based on observational evidence, levofloxacin is a reasonable alternative to TMP-SMX for the treatment of bloodstream and lower respiratory tract infections caused by S maltophilia.

Association Between Caseload Surge and COVID-19 Survival in 558 U.S. Hospitals, March to August 2020.

BACKGROUND: Several U.S. hospitals had surges in COVID-19 caseload, but their effect on COVID-19 survival rates remains unclear, especially independent of temporal changes in survival. OBJECTIVE: To determine the association between hospitals' severity-weighted COVID-19 caseload and COVID-19 mortality risk and identify effect modifiers of this relationship. DESIGN: Retrospective cohort study. (ClinicalTrials.gov: NCT04688372). SETTING: 558 U.S. hospitals in the Premier Healthcare Database. PARTICIPANTS: Adult COVID-19-coded inpatients admitted from March to August 2020 with discharge dispositions by October 2020. MEASUREMENTS: Each hospital-month was stratified by percentile rank on a surge index (a severity-weighted measure of COVID-19 caseload relative to pre-COVID-19 bed capacity). The effect of surge index on risk-adjusted odds ratio (aOR) of in-hospital mortality or discharge to hospice was calculated using hierarchical modeling; interaction by surge attributes was assessed. RESULTS: Of 144 116 inpatients with COVID-19 at 558 U.S. hospitals, 78 144 (54.2%) were admitted to hospitals in the top surge index decile. Overall, 25 344 (17.6%) died; crude COVID-19 mortality decreased over time across all surge index strata. However, compared with nonsurging (<50th surge index percentile) hospital-months, aORs in the 50th to 75th, 75th to 90th, 90th to 95th, 95th to 99th, and greater than 99th percentiles were 1.11 (95% CI, 1.01 to 1.23), 1.24 (CI, 1.12 to 1.38), 1.42 (CI, 1.27 to 1.60), 1.59 (CI, 1.41 to 1.80), and 2.00 (CI, 1.69 to 2.38), respectively. The surge index was associated with mortality across ward, intensive care unit, and intubated patients. The surge-mortality relationship was stronger in June to August than in March to May (slope difference, 0.10 [CI, 0.033 to 0.16]) despite greater corticosteroid use and more judicious intubation during later and higher-surging months. Nearly 1 in 4 COVID-19 deaths (5868 [CI, 3584 to 8171]; 23.2%) was potentially attributable to hospitals strained by surging caseload. LIMITATION: Residual confounding. CONCLUSION: Despite improvements in COVID-19 survival between March and August 2020, surges in hospital COVID-19 caseload remained detrimental to survival and potentially eroded benefits gained from emerging treatments. Bolstering preventive measures and supporting surging hospitals will save many lives. PRIMARY FUNDING SOURCE: Intramural Research Program of the National Institutes of Health Clinical Center, the National Institute of Allergy and Infectious Diseases, and the National Cancer Institute.

Manual Reading of Sensititre Broth Microdilution System Panels Improves Accuracy of Susceptibility Reporting for Polymyxin Antibiotics.

Accurate and reproducible antimicrobial susceptibility testing (AST) of polymyxin antibiotics is critical, as these drugs are last-line therapeutic options for the treatment of multidrug-resistant Gram-negative bacterial infections. However, polymyxin AST in the routine laboratory remains challenging. In this study, we evaluated the performance of an automated broth microdilution (BMD) system (Sensititre, ThermoFisher) compared to that of agar dilution (AD) for colistin and polymyxin B AST of 129 Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex clinical isolates. MICs derived from the Sensititre instrument based on two operator comparisons demonstrated overall categorical agreement (CA) of 86% and 89% compared to AD for colistin and 89% and 92% compared to AD for polymyxin B. However, error rates were higher than recommended by CLSI. Manual inspection of microdilution wells revealed microbial growth and skip wells which were erroneously interpreted by the Aris 2X instrument. Using manually interpreted BMD MICs read by two operators increased the overall categorical agreements to 88% and 95% compared to AD for colistin and 92% and 96% compared to AD for polymyxin B. Laboratories choosing to use the Sensititre platform for polymyxin AST should consider manual evaluation of wells as part of their algorithm.

Genomic insights into the host specific adaptation of the Pneumocystis genus.

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans.

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

Distinguishing disseminated Mycobacterium marinum from multifocal cutaneous disease in persons with human immunodeficiency virus/AIDS can present a diagnostic challenge, especially in the context of immune reconstitution inflammatory syndrome (IRIS). In this work, we demonstrate the utility of flow cytometry and whole genome sequencing (WGS) to diagnose disseminated M. marinum unmasked by IRIS following initiation of antiretroviral therapy. Flow cytometry demonstrated robust cytokine production by CD4 T cells in response to stimulation with M. marinum lysate. WGS of isolates from distinct lesions was consistent with clonal dissemination, supporting that preexisting disseminated M. marinum disease was uncovered by inflammatory manifestations, consistent with unmasking mycobacterial IRIS.

Effectiveness of adjunctive clindamycin in ß-lactam antibiotic-treated patients with invasive ß-haemolytic streptococcal infections in US hospitals: a retrospective multicentre cohort study.

BACKGROUND: Clindamycin is strongly recommended as an adjunctive treatment to ß-lactam antibiotics in patients with severe invasive group A ß-haemolytic streptococcal (iGAS) infections. However, there is little evidence of a benefit in the use of clindamycin in humans, and its role, if any, in treating patients with invasive non-group A/B ß-haemolytic streptococcal (iNABS) infections is unclear. METHODS: For this retrospective multicentre cohort study, we used a dataset from patients in the Cerner Health Facts database, which contains electronic health-based data from 233 US hospitals. We queried the Cerner Health Facts database for inpatients (no age restriction) admitted to hospital in 2000-15, with any clinical cultures positive for ß-haemolytic streptococcal taxa of interest, and who had received ß-lactam antibiotics within 3 days either side of culture sampling. This group of patients was then queried for those who had also received intravenous or oral clindamycin within 3 days either side of culture sampling. Patients were excluded if they had polymicrobial growth or clindamycin non-susceptible isolates, received linezolid, or had missing variable data needed for analysis. Patients were categorised by Lancefield group (iGAS or iNABS); ß-lactam antibiotic-treated patients who had received clindamycin were propensity-matched (1:2) to those who did not receive clindamycin separately for iGAS and iNABS cohorts, and logistic regression was then used to account for residual confounding factors. The primary outcome was the adjusted odds ratio (aOR) of in-hospital mortality in propensity-matched patients treated with adjunctive clindamycin versus those not treated with clindamycin in the iGAS and iNABS infection cohorts. FINDINGS: We identified 1956 inpatients with invasive ß-haemolytic streptococcal infection who had been treated with ß-lactam antibiotics across 118 hospitals (1079 with iGAS infections and 877 with iNABS infections). 459 (23·4%) of these patients had received adjunctive clindamycin treatment (343 [31·7%] patients with iGAS infections and 116 [13·2%] patients with iNABS infections). The effect of adjunctive clindamycin therapy on in-hospital mortality differed significantly and showed the opposite trend in iGAS and iNABS infection cohorts (p=0·013 for an interaction). In the iGAS cohort, in-hospital mortality in propensity-matched patients who received adjunctive clindamycin (18 [6·5%] of 277 patients) was significantly lower than in those who did not (55 [11·0%] of 500 patients; aOR 0·44 [95% CI 0·23-0·81]). This survival benefit was maintained even in patients without shock or necrotising fasciitis (six [2·6%] of 239 patients treated with adjunctive clindamycin vs 27 [6·1%] of 422 patients not treated with adjunctive clindamycin; aOR 0·40 [0·15-0·91]). By contrast, in the iNABS infection cohort, in-hospital mortality in propensity-matched patients who received adjunctive clindamycin (ten [9·8%] of 102) was higher than in those who did not (nine [4·6%] of 193), but this difference was not significant (aOR 2·60 [0·94-7·52]). Several subset analyses found qualitatively similar results. INTERPRETATION: Real-world data suggest that increased use of adjunctive clindamycin for invasive iGAS infections, but not iNABS infections, could improve outcomes, even in patients without shock or necrotising fasciitis. FUNDING: Intramural Research Program of the National Institutes of Health Clinical Center and the National Institute of Allergy and Infectious Disease.

Inappropriate empirical antibiotic therapy for bloodstream infections based on discordant in-vitro susceptibilities: a retrospective cohort analysis of prevalence, predictors, and mortality risk in US hospitals.

BACKGROUND: The prevalence and effects of inappropriate empirical antibiotic therapy for bloodstream infections are unclear. We aimed to establish the population-level burden, predictors, and mortality risk of in-vitro susceptibility-discordant empirical antibiotic therapy among patients with bloodstream infections. METHODS: Our retrospective cohort analysis of electronic health record data from 131 hospitals in the USA included patients with suspected-and subsequently confirmed-bloodstream infections who were treated empirically with systemic antibiotics between Jan 1, 2005, and Dec 31, 2014. We included all patients with monomicrobial bacteraemia caused by common bloodstream pathogens who received at least one systemic antibiotic either on the day blood cultures were drawn or the day after, and for whom susceptibility data were available. We calculated the prevalence of discordant empirical antibiotic therapy-which was defined as receiving antibiotics on the day blood culture samples were drawn to which the cultured isolate was not susceptible in vitro-overall and by hospital type by using regression tree analysis. We used generalised estimating equations to identify predictors of receiving discordant empirical antibiotic therapy, and used logistic regression to calculate adjusted odds ratios for the relationship between in-hospital mortality and discordant empirical antibiotic therapy. FINDINGS: 21 608 patients with bloodstream infections received empirical antibiotic therapy on the day of first blood culture collection. Of these patients, 4165 (19%) received discordant empirical antibiotic therapy. Discordant empirical antibiotic therapy was independently associated with increased risk of mortality (adjusted odds ratio 1·46 [95% CI, 1·28-1·66]; p<0·0001), a relationship that was unaffected by the presence or absence of resistance or sepsis or septic shock. Infection with antibiotic-resistant species strongly predicted receiving discordant empirical therapy (adjusted odds ratio 9·09 [95% CI 7·68-10·76]; p<0·0001). Most incidences of discordant empirical antibiotic therapy and associated deaths occurred among patients with bloodstream infections caused by Staphylococcus aureus or Enterobacterales. INTERPRETATION: Approximately one in five patients with bloodstream infections in US hospitals received discordant empirical antibiotic therapy, receipt of which was closely associated with infection with antibiotic-resistant pathogens. Receiving discordant empirical antibiotic therapy was associated with increased odds of mortality overall, even in patients without sepsis. Early identification of bloodstream pathogens and resistance will probably improve population-level outcomes. FUNDING: US National Institutes of Health, US Centers for Disease Control and Prevention, and US Agency for Healthcare Research and Quality.

Pharmacoepidemiology of Ceftazidime-Avibactam Use: A Retrospective Cohort Analysis of 210 US Hospitals.

BACKGROUND: Ceftazidime-avibactam has in vitro activity against some carbapenem-resistant gram-negative infections (GNIs), and therefore may be a useful alternative to more toxic antibiotics such as colistin. Understanding ceftazidime-avibactam uptake and usage patterns would inform hospital formularies, stewardship, and antibiotic development. METHODS: A retrospective cohort study assessed inpatient encounters in the Vizient database. Ceftazidime-avibactam and colistin administrations were categorized into presumed empiric (3 consecutive days of therapy or less with qualifying exclusions) versus targeted therapy (≥4 consecutive days of therapy) for presumed carbapenem-resistant GNIs. Quarterly percentage change (QPC) using modified Poisson regression and relative change in frequency of targeted ceftazidime-avibactam to colistin encounters was calculated. Factors associated with preferentially receiving targeted ceftazidime-avibactam versus colistin were identified using generalized estimating equations. RESULTS: Between 2015 quarter (q) 1 and 2017q4, ceftazidime-avibactam was administered 21 215 times across 1901 encounters. Inpatient prescriptions for ceftazidime-avibactam increased from 0.44/10 000 hospitalizations in 2015q1 to 7.7/10 000 in 2017q4 (QPC, +11%; 95% CI, 10-13%; P < .01), while conversely colistin prescriptions decreased quarterly by 5% (95% CI, 4-6%; P < .01). Ceftazidime-avibactam therapy was categorized as empiric 25% of the time, targeted 65% of the time, and indeterminate 10% of the time. Patients with chronic kidney disease were twice as likely to receive targeted ceftazidime-avibactam versus colistin (RR, 2.02; 95% CI, 1.82-2.25), whereas those on dialysis were less likely to receive ceftazidime-avibactam than colistin (RR, 0.71; 95% CI, .61-.83). CONCLUSIONS: Since approval in 2015, ceftazidime-avibactam use has grown for presumed carbapenem-resistant GNIs, while colistin has correspondingly declined. Renal function drove the choice between ceftazidime-avibactam and colistin as targeted therapy.

A Phylogeny-Informed Proteomics Approach for Species Identification within the Burkholderia cepacia Complex.

Ancestral genetic exchange between members of many important bacterial pathogen groups has resulted in phylogenetic relationships better described as networks than as bifurcating trees. In certain cases, these reticulated phylogenies have resulted in phenotypic and molecular overlap that challenges the construction of practical approaches for species identification in the clinical microbiology laboratory. Burkholderia cepacia complex (Bcc), a betaproteobacteria species group responsible for significant morbidity in persons with cystic fibrosis and chronic granulomatous disease, represents one such group where network-structured phylogeny has hampered the development of diagnostic methods for species-level discrimination. Here, we present a phylogeny-informed proteomics approach to facilitate diagnostic classification of pathogen groups with reticulated phylogenies, using Bcc as an example. Starting with a set of more than 800 Bcc and Burkholderia gladioli whole-genome assemblies, we constructed phylogenies with explicit representation of inferred interspecies recombination. Sixteen highly discriminatory peptides were chosen to distinguish B. cepacia, Burkholderia cenocepacia, Burkholderia multivorans, and B. gladioli and multiplexed into a single, rapid liquid chromatography-tandem mass spectrometry multiple reaction monitoring (LC-MS/MS MRM) assay. Testing of a blinded set of isolates containing these four Burkholderia species demonstrated 50/50 correct automatic negative calls (100% accuracy with a 95% confidence interval [CI] of 92.9 to 100%), and 70/70 correct automatic species-level positive identifications (100% accuracy with 95% CI 94.9 to 100%) after accounting for a single initial incorrect identification due to a preanalytic error, correctly identified on retesting. The approach to analysis described here is applicable to other pathogen groups for which development of diagnostic classification methods is complicated by interspecies recombination.