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.

Bacterial Genomics for National Antimicrobial Resistance Surveillance in Cambodia.

BACKGROUND: Antimicrobial resistance (AMR) surveillance in low- and middle-income countries (LMICs) often relies on poorly resourced laboratory processes. Centralized sequencing was combined with cloud-based, open-source bioinformatics solutions for national AMR surveillance in Cambodia. METHODS: Blood cultures growing gram-negative bacteria were collected at six Cambodian hospitals (January 2021 - October 2022). Isolates were obtained from pure plate growth and shotgun DNA sequencing performed in-country. Using public nucleotide and protein databases, reads were aligned for pathogen identification and AMR gene characterization. Multilocus sequence typing was performed on whole genome assemblies and haplotype clusters compared against published genomes. FINDINGS: Genes associated with acquired resistance to fluoroquinolones were identified in 59%, TMP/SMX in 45%, and aminoglycosides in 52% of 715 isolates. Extended-spectrum beta-lactamase encoding genes were identified in 34% isolates, most commonly blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55 in E. coli sequence types 131 and 1193. Carbapenemase genes were identified in 12% isolates, most commonly blaOXA-23, blaNDM-1, blaOXA-58 and blaOXA-66 in Acinetobacter species. Phylogenetic analysis revealed clonal strains of A. baumannii, representing suspected nosocomial outbreaks, and genetic clusters of quinolone-resistant typhoidal Salmonella and ESBL E. coli cases suggesting community transmission. INTERPRETATION: With accessible sequencing platforms and bioinformatics solutions, bacterial genomics can supplement AMR surveillance in LMICs. FUNDING: Research was supported by the National Institute of Allergy and Infectious Diseases and the Bill and Melinda Gates Foundation [OPP1211806].

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.

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.

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.

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.

Investigation of a Cluster of Sphingomonas koreensis Infections.

BACKGROUND: Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation. METHODS: We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions. RESULTS: The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses. CONCLUSIONS: This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.).

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.

From the Pipeline to the Bedside: Advances and Challenges in Clinical Metagenomics.

Next-generation sequencing (NGS) technologies have revolutionized multiple areas in the field of infectious diseases, from pathogen discovery to characterization of genes mediating drug resistance. Consequently, there is much anticipation that NGS technologies may be harnessed in the realm of diagnostic methods to complement or replace current culture-based and molecular microbiologic techniques. In this context, much consideration has been given to hypothesis-free, culture-independent tests that can be performed directly on primary clinical samples. The closest realizations of such universal diagnostic methods achieved to date are based on targeted amplicon and unbiased metagenomic shotgun NGS approaches. Depending on the exact details of implementation and analysis, these approaches have the potential to detect viruses, bacteria, fungi, parasites, and archaea, including organisms that were previously undiscovered and those that are uncultivatable. Shotgun metagenomics approaches additionally can provide information on the presence of virulence and resistance genetic elements. While many limitations to the use of NGS in clinical microbiology laboratories are being overcome with decreasing technology costs, expanding curated pathogen sequence databases, and better data analysis tools, there remain many challenges to the routine use and implementation of these methods. This review summarizes recent advances in applications of targeted amplicon and shotgun-based metagenomics approaches to infectious disease diagnostic methods. Technical and conceptual challenges are considered, along with expectations for future applications of these techniques.

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.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Rapid Identification of New Delhi Metallo-ß-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS.

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-ß-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/µg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

Identification of the OXA-48 Carbapenemase Family by Use of Tryptic Peptides and Liquid Chromatography-Tandem Mass Spectrometry.

Phenotypic detection of the OXA-48-type class D ß-lactamases in Enterobacteriaceae is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in subcultured bacterial isolates based on a genoproteomic approach. Following in silico trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics: ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family ß-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that included 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed including a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgment was applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2% to 100%) and 100% specificity (95% CI, 96.2% to 100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.

Rapid detection of colistin resistance protein MCR-1 by LC-MS/MS.

BACKGROUND: Colistin (polymyxin E) and polymixin B are important bactericidal antibiotics used in the treatment of serious infections caused by multi-drug resistant Gram-negative organisms. Transferrable plasmid-mediated colistin resistance, conferred by the product of the mcr-1 gene, has emerged as a global healthcare threat. Consequently, the rapid detection of the MCR-1 protein in clinical bacterial isolates has become increasingly important. We used a genoproteomic approach to identify unique peptides of the MCR-1 protein that could be detected rapidly by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: MCR-1 tryptic peptides that were efficiently ionized and readily detectable were characterized in a set of mcr-1-containing isolates with triple quadrupole LC-MS. Three optimal peptides were selected for the development of a rapid multiple reaction monitoring LC-MS/MS assay for the MCR-1 protein. To investigate the feasibility of rapid detection of the MCR-1 protein in bacterial isolates using this assay, a blinded 99-sample test set was built that included three additional mcr-1-containing clinical isolates tested in triplicate (9 samples) and 90 negative control isolates. RESULTS: All of the mcr-1-containing isolates in the test set were accurately identified with no false positive detections by three independent, blinded operators, yielding an overall performance of 100% sensitivity and specificity for multiple operators. Among the three peptides tested in this study, the best performing was DTFPQLAK. The isolate-to-result time for the assay as implemented is less than 90 min. CONCLUSIONS: This work demonstrates the feasibility of rapid detection of the MCR-1 protein in bacterial isolates by LC-MS/MS.

Compatibility of Maximum-Containment Virus-Inactivation Protocols With Identification of Bacterial Coinfections by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Diagnostics and research analyses involving samples containing maximum-containment viruses present unique challenges, and inactivation protocols compatible with downstream testing are needed. Our aim was to identify a validated viral inactivation protocol compatible with bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We assessed a panel of bacteria with 6 validated maximum-containment virus-inactivation protocols and report that inactivation with TRIzol or γ-irradiation is compatible with MALDI-TOF MS. The availability, simplicity, and rapidity of TRIzol inactivation make this method the more suitable choice.

Difficult-to-Treat Resistance in Gram-negative Bacteremia at 173 US Hospitals: Retrospective Cohort Analysis of Prevalence, Predictors, and Outcome of Resistance to All First-line Agents.

Background: Resistance to all first-line antibiotics necessitates the use of less effective or more toxic "reserve" agents. Gram-negative bloodstream infections (GNBSIs) harboring such difficult-to-treat resistance (DTR) may have higher mortality than phenotypes that allow for ≥1 active first-line antibiotic. Methods: The Premier Database was analyzed for inpatients with select GNBSIs. DTR was defined as intermediate/resistant in vitro to all ß-lactam categories, including carbapenems and fluoroquinolones. Prevalence and aminoglycoside resistance of DTR episodes were compared with carbapenem-resistant, extended-spectrum cephalosporin-resistant, and fluoroquinolone-resistant episodes using CDC definitions. Predictors of DTR were identified. The adjusted relative risk (aRR) of mortality was examined for DTR, CDC-defined phenotypes susceptible to ≥1 first-line agent, and graded loss of active categories. Results: Between 2009-2013, 471 (1%) of 45011 GNBSI episodes at 92 (53.2%) of 173 hospitals exhibited DTR, ranging from 0.04% for Escherichia coli to 18.4% for Acinetobacter baumannii. Among patients with DTR, 79% received parenteral aminoglycosides, tigecycline, or colistin/polymyxin-B; resistance to all aminoglycosides occurred in 33%. Predictors of DTR included urban healthcare and higher baseline illness. Crude mortality for GNBSIs with DTR was 43%; aRR was higher for DTR than for carbapenem-resistant (1.2; 95% confidence interval, 1.0-1.4; P = .02), extended-spectrum cephalosporin-resistant (1.2; 1.1-1.4; P = .001), or fluoroquinolone-resistant (1.2; 1.0-1.4; P = .008) infections. The mortality aRR increased 20% per graded loss of active first-line categories, from 3-5 to 1-2 to 0. Conclusion: Nonsusceptibility to first-line antibiotics is associated with decreased survival in GNBSIs. DTR is a simple bedside prognostic measure of treatment-limiting coresistance.

Severe BCG-osis Misdiagnosed as Multidrug-Resistant Tuberculosis in an IL-12Rß1-Deficient Peruvian Girl.

PURPOSE: Mendelian suceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing to severe disease caused by mycobacteria and other intracellular pathogens. Delay in diagnosis can have an impact on the patient's prognosis. METHODS: We evaluated the IFN-γ circuit by studying IFN-γ production after mycobacterial challenge as well as IL-12Rß1 expression and STAT4 phosphorylation in response to IL-12p70 stimulation in whole blood of a 6-year-old Peruvian girl with disseminated recurrent mycobacterial infection diagnosed as multidrug-resistant tuberculosis. Genetic studies with Sanger sequencing were used to identify the causative mutation. Microbiological studies based on PCR reactions were used to diagnose the specific mycobacterial species. RESULTS: We identified a homozygous mutation in the IL12RB1 gene (p. Arg211*) causing abolished expression of IL-12Rß1 and IL-12 response. MSMD diagnosis led to a microbiological reevaluation of the patient, revealing a BCG vaccine-related infection instead of tuberculosis. Treatment was then adjusted, with good response. CONCLUSIONS: We report the first Peruvian patient with IL-12Rß1 deficiency. Specific mycobacterial species diagnosis within Mycobacterium tuberculosis complex is still challenging in countries with limited access to PCR-based microbiological diagnostic techniques. Awareness of MSMD warning signs and accurate microbiological diagnosis of mycobacterial infections are of the utmost importance for optimal diagnosis and management of affected patients.

Metagenomics for Clinical Infectious Disease Diagnostics Steps Closer to Reality.

Metagenomics approaches based on shotgun next-generation sequencing hold promise for infectious disease diagnostics. Despite substantial challenges that remain, work done over the past few years justifies excitement about the potential for these approaches to transform how clinical pathogen identification and analysis are performed. In an article in this issue of the Journal of Clinical Microbiology, M. I. Ivy et al. (J Clin Microbiol 56:e00402-18, 2018, https://doi.org/10.1128/JCM.00402-18) have applied a shotgun metagenomics approach to the diagnosis of prosthetic joint infections directly from synovial fluid. The results from this work demonstrate both the potentials and challenges of this approach applied in the clinical microbiology laboratory.

Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates.

Recent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid sequencing of three clinical isolates with both MinION and Illumina MiSeq, using different library preparation methods (2D and rapid 1D) with the goal of antimicrobial resistance gene detection. We specifically evaluated the advantages of using plasmid DNA for sequencing and the value of supplementing MinION sequences with MiSeq reads for increasing assembly accuracy. Resequencing of three plasmids in a reference Klebsiella pneumoniae isolate demonstrated ∼99% accuracy of draft MinION-only assembly and >99.9% accuracy of assembly polished with MiSeq reads. Plasmid DNA sequencing of previously uncharacterized clinical extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and K. pneumoniae isolates using MinION allowed successful identification of antimicrobial resistance genes in the draft assembly corresponding to all classes of observed plasmid-based phenotypic resistance. Importantly, use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing. With a MinION-only workflow that balances accuracy against turnaround time, full annotation of plasmid resistance gene content could be obtained in under 6 h from a subcultured isolate, less time than traditional phenotypic susceptibility testing.

A Genoproteomic Approach to Detect Peptide Markers of Bacterial Respiratory Pathogens.

BACKGROUND: Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. METHODS: In silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate <1%) were identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously. RESULTS: Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS. CONCLUSIONS: This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.