Publications by Year: 2022

COVID-19 symptomology may overlap with other circulating respiratory viruses that may also cause severe disease and for which there are specific and potentially life-saving treatments. The Abbott Alinity m Resp-4-Plex assay is a multiplex PCR assay that simultaneously detects and differentiates infection with SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). We characterized its accuracy, precision, and analytical sensitivity. All were found to be robust for measures examined. In the context of sample-to-answer, near random access automation on the Alinity m platform, we believe that the Resp-4-Plex assay offers significant utility in addressing the current needs of the SARS-CoV-2 pandemic and future needs during anticipated endemic circulation of SARS-CoV-2 with other respiratory viruses.

The relationship between COVID-19 severity and viral load is unknown. Our objective was to assess the association between viral load and disease severity in COVID-19. In this single center observational study of adults with laboratory confirmed SARS-CoV-2, the first positive in-hospital nasopharyngeal swab was used to calculate the log10 copies/ml [log10 copy number (CN)] of SARS-CoV-2. Four categories based on level of care and modified sequential organ failure assessment score (mSOFA) at time of swab were determined. Median log10CN was compared between different levels of care and mSOFA quartiles. Median log10CN was compared in patients who did and did not receive influenza vaccine, and the correlation between log10CN and D-dimer was examined. We found that of 396 patients, 54.3% were male, and 25% had no major comorbidity. Hospital mortality was 15.7%. Median mSOFA was 2 (IQR 0-3). Median log10CN was 5.5 (IQR 3.3-8.0). Median log10CN was highest in non-intubated ICU patients [6.4 (IQR 4.4-8.1)] and lowest in intubated ICU patients [3.6 (IQR 2.6-6.9)] (p value < 0.01). In adjusted analyses, this difference remained significant [mean difference 1.16 (95% CI 0.18-2.14)]. There was no significant difference in log10CN between other groups in the remaining pairwise comparisons. There was no association between median log10CN and mSOFA in either unadjusted or adjusted analyses or between median log10CN in patients with and without influenza immunization. There was no correlation between log10CN and D-dimer. We conclude, in our cohort, we did not find a clear association between viral load and disease severity in COVID-19 patients. Though viral load was higher in non-intubated ICU patients than in intubated ICU patients there were no other significant differences in viral load by disease severity.

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.

A convergent, diversity-enabling total synthesis of the natural product streptothricin F has been achieved. Herein, we describe the potent antimicrobial activity of streptothricin F and highlight the importance of a total synthesis that allows for the installation of practical divergent steps for medicinal chemistry exploits. Key features of our synthesis include a Burgess reagent-mediated 1,2-anti-diamine installation, diastereoselective azidation of a lactam enolate, and a mercury(ii) chloride-mediated desulfurization-guanidination. The development of this chemistry enables the synthesis and structure-activity studies of streptothricin F analogs.

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.

Clinical Microbiology Open (CMO), a meeting supported by the American Society for Microbiology's Clinical and Public Health Microbiology Committee (CPHMC) and Corporate Council, provides a unique interactive platform for leaders from diagnostic microbiology laboratories, industry, and federal agencies to discuss the current and future state of the clinical microbiology laboratory. The purpose is to leverage the group's diverse views and expertise to address critical challenges, and discuss potential collaborative opportunities for diagnostic microbiology, through the utilization of varied resources. The first and second CMO meetings were held in 2018 and 2019, respectively. Discussions were focused on the diagnostic potential of innovative technologies and laboratory diagnostic stewardship, including expansion of next-generation sequencing into clinical diagnostics, improvement and advancement of molecular diagnostics, emerging diagnostics, including rapid antimicrobial susceptibility and point of care testing (POCT), harnessing big data through artificial intelligence, and staffing in the clinical microbiology laboratory. Shortly after CMO 2019, the coronavirus disease 2019 (COVID-19) pandemic further highlighted the need for the diagnostic microbiology community to work together to utilize and expand on resources to respond to the pandemic. The issues, challenges, and potential collaborative efforts discussed during the past two CMO meetings proved critical in addressing the COVID-19 response by diagnostic laboratories, industry partners, and federal organizations. Planning for a third CMO (CMO 2022) is underway and will transition from a discussion-based meeting to an action-based meeting. The primary focus will be to reflect on the lessons learned from the COVID-19 pandemic and better prepare for future pandemics.

The development and application of the asymmetric synthesis of oligosaccharides from achiral starting materials is reviewed. This de novo asymmetric approach centers around the use of asymmetric catalysis for the synthesis of optically pure furan alcohols in conjunction with Achmatowicz oxidative rearrangement for the synthesis of various pyranones. In addition, the use of a diastereoselective palladium-catalyzed glycosylation and subsequent diastereoselective post-glycosylation transformation was used for the synthesis of oligosaccharides. The application of this approach to oligosaccharide synthesis is discussed.

Implementing effective antimicrobial therapy close to the onset of infection lowers morbidity and mortality and attenuates the spread of antimicrobial resistance. Current antimicrobial susceptibility testing (AST) methods, however, require several days to determine optimal therapies. We present technology and an automated platform that identify (ID) Urinary Tract Infection pathogens in 45 min and provide phenotypic AST results in less than 5 h from urine specimens without colony isolation. The ID and AST tests count cells fluorescently labeled with specific rRNA probes using non-magnified digital imaging. The ID test detected five pathogens at ≤ 7,000 CFU/mL and had a linear range of   4 orders of magnitude. For contrived specimens, AST tests gave 93.1% categorical agreement with 1.3% Very Major Errors (VME), 0.3% Major Errors (ME), and 6.3% minor Errors (mE) compared to the broth microdilution (BMD) reference method. For clinical specimens, the ID test had 98.6% agreement and the AST test had 92.3% categorical agreement with 4.2% mE, 3.4% ME and 4.0% VME compared to BMD. Data presented demonstrates that direct-from-specimen AST tests can accurately determine antimicrobial susceptibility/resistance for each pathogen in a specimen containing two pathogens. The method is robust to urine matrix effects and off-target commensal and contaminating bacteria.