Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. METHODS: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years. RESULTS: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. CONCLUSIONS: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

Transcriptomics Reveals How Minocycline-Colistin Synergy Overcomes Antibiotic Resistance in Multidrug-Resistant Klebsiella pneumoniae.

Multidrug-resistant Gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using transcriptome sequencing (RNA-Seq), we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.

Evaluation of the Synergistic Activity of Antibacterial and Antifungal Drugs against Candida auris Using an Inkjet Printer-Assisted Method.

Candida auris is an emerging multidrug-resistant fungal pathogen that spreads readily in health care settings and has caused numerous hospital outbreaks. Very few treatment options exist for C. auris infections. We evaluated the activity of all two-drug combinations of three antifungal agents (amphotericin B, caspofungin, and voriconazole) and two antibacterial agents (minocycline and rifampin) against a collection of 10 C. auris isolates using an automated, inkjet printer-assisted checkerboard array method. Three antibacterial-antifungal combinations (amphotericin B plus rifampin, amphotericin B plus minocycline, and caspofungin plus minocycline) demonstrated synergistic activity by checkerboard array against ≥90% of strains, with fractional inhibitory concentration index (FICI) values of 0.094 to 0.5. The two amphotericin B-containing combinations were also synergistic using the time-kill synergy testing method, with up to a 4.99-log10 decrease in surviving yeast compared to either agent alone. Our results suggest that combinations of antifungal and antibacterial agents provide a promising avenue for treatment of this multidrug-resistant pathogen.

New strategies and structural considerations in development of therapeutics for carbapenem-resistant Enterobacteriaceae.

Antimicrobial resistance poses a significant threat to our ability to treat infections. Especially concerning is the emergence of carbapenem-resistant Enterobacteriaceae (CRE). In the new 2019 United States Centers for Disease Control and Prevention Antibiotic Resistance Report, CRE remain in the most urgent antimicrobial resistance threat category. There is good reason for this concerning designation. In particular, the combination of several resistance elements in CRE can make these pathogens untreatable or effectively untreatable with our current armamentarium of anti-infective agents. This article reviews recently approved agents with activity against CRE and a range of modalities in the pipeline, from early academic investigation to those in clinical trials, with a focus on structural aspects of new antibiotics. Another article in this series addresses the need to incentive pharmaceutical companies to invest in CRE antimicrobial development and to encourage hospitals to make these agents available in their formularies. This article will also consider the need for change in requirements for antimicrobial susceptibility testing implementation in clinical laboratories to address practical roadblocks that impede our efforts to provide even existing CRE antibiotics to our patients.

Bringing Antimicrobial Susceptibility Testing for New Drugs into the Clinical Laboratory: Removing Obstacles in Our Fight against Multidrug-Resistant Pathogens.

There are now several new antibiotics available to treat multidrug-resistant pathogens, and susceptibility testing methods for these drugs are increasingly available at the time of drug approval. However, lack of clarity regarding verification requirements remains a formidable barrier to introducing such testing in clinical laboratories, making these drugs practically unavailable for patient use. We propose a change in the framework for bringing in testing for new antibiotics, focusing on quality control rather than underpowered verification studies.

When One Drug Is Not Enough: Context, Methodology, and Future Prospects in Antibacterial Synergy Testing.

Antibacterial combinations have long been used to accomplish a variety of therapeutic goals, including prevention of resistance and enhanced antimicrobial activity. In vitro synergy testing methods, including the checkerboard array, the time-kill study, diffusion assays, and pharmacokinetic/pharmacodynamic models, are used commonly in the research setting, but are not routinely performed in the clinical microbiology laboratory because of test complexity and uncertainty about their predictive value for patient outcomes. Optimized synergy testing techniques and better data on the relationship between in vitro results and clinical outcomes are needed to guide the rational use of antimicrobial combinations in the multidrug resistance era.

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method.

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing. The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Synergistic Activity of Colistin-Containing Combinations against Colistin-Resistant Enterobacteriaceae.

Resistance to colistin, a polypeptide drug used as an agent of last resort for the treatment of infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE), severely limits treatment options and may even transform an XDR organism into one that is pan-resistant. We investigated the synergistic activity of colistin in combination with 19 antibiotics against a collection of 20 colistin-resistant Enterobacteriaceae isolates, 15 of which were also CRE. All combinations were tested against all strains using an inkjet printer-assisted digital dispensing checkerboard array, and the activities of those that demonstrated synergy by this method were evaluated against a single isolate in a time-kill synergy study. Eighteen of 19 combinations demonstrated synergy against two or more isolates, and the 4 most highly synergistic combinations (colistin combined with linezolid, rifampin, azithromycin, and fusidic acid) were synergistic against ≥90% of strains. Sixteen of 18 combinations (88.9%) that were synergistic in the checkerboard array were also synergistic in a time-kill study. Our findings demonstrate that colistin in combination with a range of antibiotics, particularly protein and RNA synthesis inhibitors, exhibits synergy against colistin-resistant strains, suggesting that colistin may exert a subinhibitory permeabilizing effect on the Gram-negative bacterial outer membrane even in isolates that are resistant to it. These findings suggest that colistin combination therapy may have promise as a treatment approach for patients infected with colistin-resistant XDR Gram-negative pathogens.

Evaluation of apramycin activity against methicillin-resistant, methicillin-sensitive, and vancomycin-intermediate Staphylococcus aureus clinical isolates.

We evaluated the in vitro activity of apramycin against clinical strains of vancomycin-intermediate and methicillin-resistant and -susceptible Staphylococcus aureus. Apramycin demonstrated an MIC50/MIC90 of 8/16 µg/mL. No strains had an MIC above the epidemiological cutoff value of 32 µg/mL, suggesting apramycin resistance mechanisms are rare in this strain population. The mounting evidence for broad-spectrum in vitro activity of apramycin against S. aureus and other bacterial species suggests that further exploration of apramycin or derivatives as repurposed human therapeutics is warranted.

Adherence to guidelines for testing and treatment of children with pharyngitis: a retrospective study.

BACKGROUND: Group A streptococcus (GAS) is the most common bacterial etiology of pharyngitis but is difficult to distinguish clinically from viral pharyngitis. There are benefits to early antibacterial treatment of GAS pharyngitis, but administering antibiotics to children with viral pharyngitis is ineffective and costly. We evaluated adherence to guidelines that were developed to help clinicians distinguish between viral and GAS pharyngitis and guide management. METHODS: Retrospective cohort study of patients ages 3-18 who had a rapid streptococcal test and/or throat culture performed in an outpatient setting. We collected data on documentation of components of the McIsaac score and classified tests as indicated if the score was ≥2. Based on McIsaac score and GAS test results, we determined whether each antibiotic course prescribed was indicated according to the Infectious Diseases Society of America guideline. RESULTS: Among 291 eligible children, 87 (30%) had all five components of the McIsaac score documented. There was sufficient data to classify the score as either < 2 or ≥2 in 234 (80%); among these, 96% of tests were indicated. Twenty-nine patients (10%) were prescribed antibiotics. Eight (28%) of these prescriptions were not indicated according to guidelines. CONCLUSIONS: The majority of GAS tests in children with pharyngitis are indicated, although providers do not regularly document all elements of a validated pharyngitis scoring tool. Over one quarter of children prescribed antibiotics for pharyngitis did not require antibiotics according to guidelines. There remains a role for targeted antimicrobial stewardship education regarding pharyngitis management in pediatric outpatient settings.

Arthroconidia in lung tissue: an unusual histopathological finding in pulmonary coccidioidomycosis.

Coccidioides immitis/posadasii presents in mycelial form with branching hyphae and arthroconidia when cultured in the laboratory. On histopathology, the presence of endospore-containing spherules is considered diagnostic of coccidioidomycosis. Here we report an unusual case of coccidioidomycosis with hyphae and arthroconidia in pulmonary tissue sections. A 49-year-old male patient with intermittently treated pulmonary coccidioidomycosis sought treatment for residual pulmonary complaints. A cavity in the left upper lobe was seen on computed tomographic scan. Due to minimal improvement of symptoms despite treatment with fluconazole, a left upper lobectomy was ultimately performed. Coccidioides mimmitis/posadasii was identified by culture and DNA probe from the lobectomy specimen. The histopathology showed a fibro-cavitary lesion, with arthroconidia and hyphal structures, but no typical endospore-forming spherules. While uncommon, C. immitis/posadasii may present with hyphae and arthroconidia on histopathology. Pathologists should be aware of this unusual presentation; culture remains the most reliable method for definitive diagnosis.

Screening for synergistic activity of antimicrobial combinations against carbapenem-resistant Enterobacteriaceae using inkjet printer-based technology.

Background: Synergistic combination antimicrobial therapy may provide new options for treatment of MDR infections. However, comprehensive in vitro synergy data are limited and facile methods to perform synergy testing in a clinically actionable time frame are unavailable. Objectives: To systematically investigate a broad range of antibiotic combinations for evidence of synergistic activity against a collection of carbapenem-resistant Enterobacteriaceae (CRE) isolates. Methods: We made use of an automated method for chequerboard array synergy testing based on inkjet printer technology in the HP D300 digital dispenser to test 56 pairwise antimicrobial combinations of meropenem, aztreonam, cefepime, colistin, gentamicin, levofloxacin, chloramphenicol, fosfomycin, trimethoprim/sulfamethoxazole, minocycline and rifampicin, as well as the double carbapenem combination of meropenem and ertapenem. Results: In a screening procedure, we tested these combinations against four CRE strains and identified nine antibiotic combinations that showed potential clinically relevant synergy. In confirmatory testing using 10 CRE strains, six combinations demonstrated clinically relevant synergy with both antimicrobials at the minimum fractional inhibitory concentration (FICI-MIN) in the susceptible or intermediate range in at least one trial. These included two novel combinations: minocycline plus colistin and minocycline plus meropenem. In 80% of strains at least one combination demonstrated clinically relevant synergy, but the combinations that demonstrated synergy varied from strain to strain. Conclusions: This work establishes the foundation for future systematic, broad-range investigations into antibiotic synergy for CRE, emphasizes the need for individualized synergy testing and demonstrates the utility of inkjet printer-based technology for the performance of automated antimicrobial synergy assays.

Development of MAST: A Microscopy-Based Antimicrobial Susceptibility Testing Platform.

Antibiotic resistance is compromising our ability to treat bacterial infections. Clinical microbiology laboratories guide appropriate treatment through antimicrobial susceptibility testing (AST) of patient bacterial isolates. However, increasingly, pathogens are developing resistance to a broad range of antimicrobials, requiring AST of alternative agents for which no commercially available testing methods are available. Therefore, there exists a significant AST testing gap in which current methodologies cannot adequately address the need for rapid results in the face of unpredictable susceptibility profiles. To address this gap, we developed a multicomponent, microscopy-based AST (MAST) platform capable of AST determinations after only a 2 h incubation. MAST consists of a solid-phase microwell growth surface in a 384-well plate format, inkjet printing-based application of both antimicrobials and bacteria at any desired concentrations, automated microscopic imaging of bacterial replication, and a deep learning approach for automated image classification and determination of antimicrobial minimal inhibitory concentrations (MICs). In evaluating a susceptible strain set, 95.8% were within ±1 and 99.4% were within ±2, twofold dilutions, respectively, of reference broth microdilution MIC values. Most (98.3%) of the results were in categorical agreement. We conclude that MAST offers promise for rapid, accurate, and flexible AST to help address the antimicrobial testing gap.

The Poisoned Well: Enhancing the Predictive Value of Antimicrobial Susceptibility Testing in the Era of Multidrug Resistance.

Antimicrobial susceptibility testing (AST) is a fundamental mission of the clinical microbiology laboratory. Reference AST methods are based on bacterial growth in antibiotic doubling dilution series, which means that any error in the reference method inherently represents at least a 2-fold difference. We describe the origins of current AST reference methodology, highlight the sources of AST variability, and propose ideas for improving AST predictive power.

Improved Accuracy of Cefepime Susceptibility Testing for Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae with an On-Demand Digital Dispensing Method.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae generally cannot be treated with penicillins and cephalosporins. However, some later-generation cephalosporins, including cefepime, are poorly hydrolyzed by specific ESBL enzymes, and certain strains demonstrate in vitro susceptibility to these agents, potentially affording additional treatment opportunities. Moreover, the ability to adjust both the dose and dosing interval of beta-lactam agents allows the treatment of strains with elevated MICs that were formerly classified in the intermediate range. The ability to treat strains with elevated cefepime MICs is codified in new susceptible dose-dependent (SDD) breakpoints promulgated by the Clinical and Laboratory Standards Institute. In the interest of validating and implementing new cefepime SDD criteria, we evaluated the performances of Vitek 2, disk diffusion, and a MicroScan panel compared to that of reference broth microdilution (BMD) during the testing of 64 strains enriched for presumptive ESBL phenotype (based on nonsusceptibility to ceftriaxone). Surprisingly, categorical agreement with BMD was only 47.6%, 57.1%, and 44.6% for the three methods, respectively. Given these findings, we tested the performance of the HP D300 inkjet-assisted broth microdilution digital dispensing method (DDM), which was previously described by our group as an at-will testing alternative. In contrast to commercial methods, DDM results correlated well with the reference method, with 86% categorical agreement, 91.1% evaluable essential agreement, and no major or very major errors. The reproducibility and accuracy of MIC determinations were statistically equivalent to BMD. Our results provide support for the use of the DDM as a BMD equivalent methodology that will enable hospital-based clinical laboratories to support cefepime MIC-based dosing strategies.

Glial vascular degeneration in CADASIL.

CADASIL is a genetic vascular dementia caused by mutations in the Notch 3 gene on Chromosome 19. However, little is known about the mechanisms of vascular degeneration. We characterized upstream components of Notch signaling pathways that may be disrupted in CADASIL, by measuring expression of insulin, IGF-1, and IGF-2 receptors, Notch 1, Notch 3, and aspartyl-(asparaginyl)-ß-hydroxylase (AAH) in cortex and white matter from 3 CADASIL and 6 control brains. We assessed CADASIL-associated cell loss by measuring mRNA corresponding to neurons, oligodendroglia, and astrocytes, and indices of vascular degeneration by measuring smooth muscle actin (SMA) and endothelin-1 expression in isolated vessels. Immunohistochemical staining was used to assess SMA degeneration. Significant abnormalities, including reduced cerebral white matter mRNA levels of Notch 1, Notch 3, AAH, SMA, IGF receptors, myelin-associated glycoproteins, and glial fibrillary acidic protein, and reduced vascular expression of SMA, IGF receptors, Notch 1, and Notch 3 were detected in CADASIL-lesioned brains. In addition, we found CADASIL-associated reductions in SMA, and increases in ubiquitin immunoreactivity in the media of white matter and meningeal vessels. No abnormalities in gene expression or immunoreactivity were observed in CADASIL cerebral cortex. In conclusion, molecular abnormalities in CADASIL are largely restricted to white matter and white matter vessels, corresponding to the distribution of neuropathological lesions. These preliminary findings suggest that CADASIL is mediated by both glial and vascular degeneration with reduced expression of IGF receptors and AAH, which regulate Notch expression and function.