The emergence and spread of antibiotic-resistant Gram-negative bacteria (rGNB) across global healthcare networks presents a significant threat to public health. As the number of effective antibiotics available to treat these resistant organisms dwindles, it is essential that we devise more effective strategies for controlling their proliferation. Recently, whole-genome sequencing has emerged as a disruptive technology that has transformed our understanding of the evolution and epidemiology of diverse rGNB species, and it has the potential to guide strategies for controlling the evolution and spread of resistance. Here, we review specific areas in which genomics has already made a significant impact, including outbreak investigations, regional epidemiology, clinical diagnostics, resistance evolution, and the study of epidemic lineages. While highlighting early successes, we also point to the next steps needed to translate this technology into strategies to improve public health and clinical medicine.

A genomic epidemiologic investigation of a putative carbapenem-resistant Enterobacter cloacae outbreak revealed few plausible instances of nosocomial transmission, highlighting instead the frequent importation of E. cloacae into our hospital. Searching for genetic determinants of carbapenem resistance demonstrated that most resistance is due to convergent mutations in phylogenetically diverse E. cloacae.

Genomic sequencing of SARS-CoV-2 continues to provide valuable insight into the ever-changing variant makeup of the COVID-19 pandemic. More than three million SARS-COV-2 genomes have been deposited in GISAID, but contributions from the United States, particularly through 2020, lagged behind the global effort. The primary goal of clinical microbiology laboratories is seldom rooted in epidemiologic or public health testing and many labs do not contain in-house sequencing technology. However, we recognized the need for clinical microbiologists to lend expertise, share specimen resources, and partner with academic laboratories and sequencing cores to assist in SARS-COV-2 epidemiologic sequencing efforts. Here we describe two clinical and academic laboratory collaborations for SARS-COV-2 genomic sequencing. We highlight roles of the clinical microbiologists and the academic labs, outline best practices, describe two divergent strategies in accomplishing a similar goal, and discuss the challenges with implementing and maintaining such programs.

The COVID-19 pandemic is complicated by cases of vaccine-breakthrough, re-infection, and widespread transmission of variants of concern (VOC). Consequently, the need to interpret longitudinal positive SARS-CoV-2 (SCV-2) tests is crucial in guiding clinical decisions regarding infection control precautions and treatment. Although quantitative tests are not routinely used diagnostically, standard diagnostic RT-PCR tests yield Ct values that are inversely correlated with RNA quantity. In this study, we performed a retrospective review of 72,217 SCV-2 PCR positive tests and identified 264 patients with longitudinal positivity prior to vaccination and VOC circulation. Patients with longitudinal positivity fell into two categories: short-term (207, 78%) or prolonged (57, 22%) positivity, defined as <= 28 (range 1-28, median 16) days and >28 (range 29- 152, median 41) days, respectively. In general, Ct values declined over time in both groups; however, 11 short-term positive patients had greater amounts of RNA detected at their terminal test compared to the first positive, and 5 patients had RNA detected at Ct < 35 at least 40 days after initial infection.  Oscillating positive and negative results occurred in both groups, although oscillation was seen three times more frequently in prolonged-positive patients. Patients with prolonged positivity had diverse clinical characteristics but were often critically ill and were discharged to high-level care or deceased (22%). Overall, this study demonstrates that caution must be emphasized when interpreting Ct values as a proxy for infectivity, predictor of severity, or a guide for patient care decisions in the absence of additional clinical context.

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Objective: Cohorting patients who are colonized or infected with multidrug-resistant organisms (MDROs) has been demonstrated to protect uncolonized patients from acquiring MDROs in healthcare settings. A neglected aspect of cohorting is the potential for cross-transmission within the cohort and the possibility of colonized patients acquiring secondary isolates with additional antibiotic resistance traits. We searched for evidence of cross-transmission of KPC+ Klebsiella pneumoniae (KPC-Kp) colonization among cohorted patients in a long-term acute care hospital (LTACH), and evaluated the impact of secondary acquisitions on resistance potential. Design: Genomic epidemiological investigation Setting: A high-prevalence LTACH during a bundled intervention that included cohorting KPC-Kp-positive patients. Methods: Whole-genome sequencing (WGS) and location data were analyzed to identify potential cases of cross-transmission between cohorted patients. Results: Secondary KPC-Kp isolates from 19 of 28 admission-positive patients were more closely related to another patient’s isolate than to their own admission isolate. In 14 of these 19 cases there was strong genomic evidence for cross-transmission (<10 SNVs) and the majority of these patients occupied shared cohort floors (12 cases) or rooms (5 cases) at the same time. Of the 14 patients with strong genomic evidence of acquisition, 12 acquired antibiotic resistance genes not found in their primary isolates. Conclusions: Acquisition of secondary KPC-Kp isolates carrying distinct antibiotic resistance genes was detected in nearly half of cohorted patients. These results highlight the importance of healthcare provider adherence to infection prevention protocols within cohort locations, and motivate future studies to assess whether multiple-strain acquisition increases risk of adverse patient outcomes.

Background Carbapenem-resistant Enterobacteriaceae have been recognized as an urgent antibiotic resistance threat for more than a decade. Despite this attention, their prevalence has remained steady or increased in some settings, suggesting that transmission pathways remain uncontrolled by current prevention strategies. We hypothesized that these transmission pathways, and hence targets for improved prevention, could be elucidated through comprehensive patient sampling, followed by integration of whole-genome sequencing (WGS) and epidemiological data. Methods Longitudinal KPC+ Klebsiella pneumoniae (KPC-Kp) surveillance cultures were collected from 94% of patients in a long-term acute care hospital (LTACH) during a one-year bundled intervention to reduce KPC-Kp prevalence. WGS of 462 KPC-Kp isolates from 256 patients, and associated surveillance data were integrated using a distance threshold-free approach to identify transmission clusters that grouped patients acquiring KPC-Kp in the LTACH with the admission-positive "index" patients that imported their strain into the facility. Plausible transmission pathways within clusters were identified using patient location data. Findings Transmission clusters (N=49) had between 2-14 patients, capturing KPC-Kp acquisitions from 100 (80%) patients who first acquired KPC-Kp in the LTACH. Within-cluster genetic diversity varied from 0-154 (median 9) single-nucleotide variants (SNVs), with elevated diversity being driven by prolonged asymptomatic colonization and evolution of hypermutator strains. Transmission between patients in clusters could be explained by spatiotemporal overlap in patient rooms (14%), wards (66%), or facility (81%). Sequential exposure to the same patient room was the only epidemiological link for one patient, indicating that residual environmental contamination of rooms after patient discharge contributed little to transmission. Persistent, modifiable routes of transmission were associated with lapses in patient cohorting, transmission between cohort and non-cohort locations and clusters propagating due to false-negative surveillance. Interpretation Integration of comprehensive surveillance and WGS data using a SNV threshold-free approach disclosed specific instances where improved patient and healthcare worker cohorting, reducing exposures to common locations outside of patient rooms, and improved KPC-Kp colonization detection could reduce transmission. Overall, results highlight the potential for WGS to monitor and improve infection prevention and the importance of combining rigorous sampling with appropriate analytical strategies to generate actionable hypotheses.