Harnessing the Diversity of Burkholderia spp. Prophages for Therapeutic Potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here, we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested phages were tested for lytic activity against the same 32 isolates. Temperate phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that Burkholdera cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested prophages were tested for lytic activity against the same 32 isolates. Lytic phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes, and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that B. cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Real-time genomic epidemiologic investigation of a multispecies plasmid-associated hospital outbreak of NDM-5-producing Enterobacterales infections.

OBJECTIVES: New Delhi metallo-ß-lactamase (NDM) is an emergent mechanism of carbapenem resistance associated with high mortality and limited treatment options. Because the blaNDM resistance gene is often carried on plasmids, traditional infection prevention and control (IP&C) surveillance methods and reactive whole genome sequencing (WGS) may not detect plasmid transfer in multispecies outbreaks. METHODS: Initial outbreak detection of NDM-producing Enterobacterales identified at an acute care hospital occurred via traditional IP&C methods and was supplemented by real-time WGS surveillance performed weekly. To resolve NDM-encoding plasmids, we performed long-read sequencing and constructed hybrid assemblies. WGS data for suspected outbreaks was shared with the IP&C team for assessment and intervention. RESULTS: We observed a multispecies outbreak of NDM-5-producing Enterobacterales isolated from 15 patients between February 2021 and February 2023. The 19 clinical and surveillance isolates sequenced included 7 bacterial species encoding the same NDM-5 plasmid. WGS surveillance and epidemiologic investigation characterized 10 horizontal plasmid transfer events and 6 bacterial transmission events between patients in varying hospital units. CONCLUSIONS: Our investigation revealed a complex, multispecies outbreak of NDM involving multiple plasmid transfer and bacterial transmission events. We highlight the utility of combining traditional IP&C and prospective genomic methods in identifying and containing plasmid-associated outbreaks.

In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

Two Artificial Tears Outbreak-Associated Cases of Extensively Drug-Resistant Pseudomonas aeruginosa Detected Through Whole Genome Sequencing-Based Surveillance.

We describe 2 cases of extensively drug-resistant Pseudomonas aeruginosa infection caused by a strain of public health concern, as it was recently associated with a nationwide outbreak of contaminated artificial tears. Both cases were detected through database review of genomes in the Enhanced Detection System for Hospital-Associated Transmission (EDS-HAT), a routine genome sequencing-based surveillance program. We generated a high-quality reference genome for the outbreak strain from an isolate from our center and examined the mobile elements encoding blaVIM-80 and bla-GES-9 carbapenemases. We used publicly available Pseudomonas aeruginosa genomes to explore the genetic relatedness and antimicrobial resistance genes of the outbreak strain.

The utility of whole-genome sequencing to inform epidemiologic investigations of SARS-CoV-2 clusters in acute-care hospitals.

OBJECTIVE: To evaluate the utility of selective reactive whole-genome sequencing (WGS) in aiding healthcare-associated cluster investigations. DESIGN: Mixed-methods quality-improvement study. SETTING: Thes study was conducted across 8 acute-care facilities in an integrated health system. METHODS: We analyzed healthcare-associated coronavirus disease 2019 (COVID-19) clusters between May 2020 and July 2022 for which facility infection prevention and control (IPC) teams selectively requested reactive WGS to aid the epidemiologic investigation. WGS was performed with real-time results provided to IPC teams, including genetic relatedness of sequenced isolates. We conducted structured interviews with IPC teams on the informativeness of WGS for transmission investigation and prevention. RESULTS: In total, 8 IPC teams requested WGS to aid the investigation of 17 COVID-19 clusters comprising 226 cases and 116 (51%) sequenced isolates. Of these, 16 (94%) clusters had at least 1 WGS-defined transmission event. IPC teams hypothesized transmission pathways in 14 (82%) of 17 clusters and used data visualizations to characterize these pathways in 11 clusters (65%). The teams reported that in 15 clusters (88%), WGS identified a transmission pathway; the WGS-defined pathway was not one that was predicted by epidemiologic investigation in 7 clusters (41%). WGS changed the understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in 8 clusters (47%) and altered infection prevention interventions in 8 clusters (47%). CONCLUSIONS: Selectively utilizing reactive WGS helped identify cryptic SARS-CoV-2 transmission pathways and frequently changed the understanding and response to SARS-CoV-2 outbreaks. Until WGS is widely adopted, a selective reactive WGS approach may be highly impactful in response to healthcare-associated cluster investigations.

Mutations in ompK36 differentially impact in vitro synergy of meropenem/vaborbactam and ceftazidime/avibactam in combination with other antibiotics against KPC-producing Klebsiella pneumoniae.

Objectives: Ceftazidime/avibactam and meropenem/vaborbactam are preferred agents for Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) infections and are often used in combination with other agents. We aimed to characterize the synergy of combinations against KPC-Kp with varying ompK36 genotypes. Methods: KPC-Kp that harboured ompK36 WT, IS5 or glycine-aspartic acid duplication (GD) genotypes were selected. MICs were determined in triplicate. Synergy was assessed by time-kill assays for ceftazidime/avibactam and meropenem/vaborbactam in combination with colistin, gentamicin, tigecycline, meropenem or fosfomycin against 1 × 108 cfu/mL KPC-Kp. Results: KPC-Kp harboured ompK36 WT (n = 5), IS5 (n = 5) or GD (n = 5); 11 were KPC-2 and 4 were KPC-3. All were susceptible to ceftazidime/avibactam and meropenem/vaborbactam. In time-kill analysis, ceftazidime/avibactam and meropenem/vaborbactam 1 × MIC exhibited mean 24 h log-kills of -2.01 and -0.84, respectively. Ceftazidime/avibactam was synergistic in combination with colistin independent of ompK36 genotype. Ceftazidime/avibactam combinations impacted by porin mutations (compared to WT) were meropenem (-5.18 versus -6.62 mean log-kill, P < 0.001) and fosfomycin (-3.98 versus -6.58, P = 0.058). Mean log-kills with meropenem/vaborbactam were greatest in combination with gentamicin (-5.36). In the presence of porin mutations, meropenem/vaborbactam killing activity was potentiated by the addition of colistin (-6.65 versus -0.70, P = 0.03) and fosfomycin (-3.12 versus 1.54, P = 0.003). Conclusions: Our results shed new light on the synergy of ceftazidime/avibactam and meropenem/vaborbactam combinations against KPC-Kp with or without porin mutations. Killing activity of ceftazidime/avibactam with other cell wall active agents was decreased against isolates with porin mutations. On the other hand, some meropenem/vaborbactam combinations demonstrated enhanced killing in the presence of porin mutations.

Genomic Epidemiologic Investigation of a Multispecies Hospital Outbreak of NDM-5-Producing Enterobacterales Infections.

Background: New Delhi metallo-ß-lactamase (NDM) represents an emergent mechanism of carbapenem resistance associated with high mortality and limited antimicrobial treatment options. Because the blaNDM resistance gene is often carried on plasmids, traditional infection prevention and control (IP&C) surveillance methods like speciation, antimicrobial resistance testing, and reactive whole genome sequencing (WGS) may not detect plasmid transfer in multispecies outbreaks. Methods: Initial outbreak detection of NDM-producing Enterobacterales identified at an acute care hospital occurred via traditional IP&C methods and was supplemented by real-time WGS surveillance, which was performed weekly using the Illumina platform. To resolve NDM-encoding plasmids, we performed long-read Oxford Nanopore sequencing and constructed hybrid assemblies using Illumina and Nanopore sequencing data. Reports of relatedness between NDM-producing organisms and reactive WGS for suspected outbreaks were shared with the IP&C team for assessment and intervention. Findings: We observed a multispecies outbreak of NDM-5-producing Enterobacterales isolated from 15 patients between February 2021 and February 2023. The 19 clinical and surveillance isolates sequenced included seven bacterial species and each encoded the same NDM-5 plasmid, which showed high homology to NDM plasmids previously observed in Asia. WGS surveillance and epidemiologic investigation characterized ten horizontal plasmid transfer events and six bacterial transmission events between patients housed in varying hospital units. Transmission prevention focused on enhanced observation and adherence to basic infection prevention measures. Interpretation: Our investigation revealed a complex, multispecies outbreak of NDM that involved multiple plasmid transfer and bacterial transmission events, increasing the complexity of outbreak identification and transmission prevention. Our investigation highlights the utility of combining traditional IP&C and prospective genomic methods in identifying and containing plasmid-associated outbreaks. Funding: This work was funded in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) (R01AI127472) (R21AI1783691).

In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

Empirically derived sequence similarity thresholds to study the genomic epidemiology of plasmids shared among healthcare-associated bacterial pathogens.

BACKGROUND: Healthcare-associated bacterial pathogens frequently carry plasmids that contribute to antibiotic resistance and virulence. The horizontal transfer of plasmids in healthcare settings has been previously documented, but genomic and epidemiologic methods to study this phenomenon remain underdeveloped. The objectives of this study were to apply whole-genome sequencing to systematically resolve and track plasmids carried by nosocomial pathogens in a single hospital, and to identify epidemiologic links that indicated likely horizontal plasmid transfer. METHODS: We performed an observational study of plasmids circulating among bacterial isolates infecting patients at a large hospital. We first examined plasmids carried by isolates sampled from the same patient over time and isolates that caused clonal outbreaks in the same hospital to develop thresholds with which horizontal plasmid transfer within a tertiary hospital could be inferred. We then applied those sequence similarity thresholds to perform a systematic screen of 3074 genomes of nosocomial bacterial isolates from a single hospital for the presence of 89 plasmids. We also collected and reviewed data from electronic health records for evidence of geotemporal links between patients infected with bacteria encoding plasmids of interest. FINDINGS: Our analyses determined that 95% of analyzed genomes maintained roughly 95% of their plasmid genetic content and accumulated fewer than 15 SNPs per 100 kb of plasmid sequence. Applying these similarity thresholds to identify horizontal plasmid transfer identified 45 plasmids that potentially circulated among clinical isolates. Ten highly preserved plasmids met criteria for geotemporal links associated with horizontal transfer. Several plasmids with shared backbones also encoded different additional mobile genetic element content, and these elements were variably present among the sampled clinical isolate genomes. INTERPRETATION: Evidence suggests that the horizontal transfer of plasmids among nosocomial bacterial pathogens appears to be frequent within hospitals and can be monitored with whole genome sequencing and comparative genomics approaches. These approaches should incorporate both nucleotide identity and reference sequence coverage to study the dynamics of plasmid transfer in the hospital. FUNDING: This research was supported by the US National Institute of Allergy and Infectious Disease (NIAID) and the University of Pittsburgh School of Medicine.

Two artificial tears outbreak-associated cases of XDR Pseudomonas aeruginosa detected through whole genome sequencing-based surveillance.

We describe two cases of XDR Pseudomonas aeruginosa infection caused by a strain of public health concern recently associated with a nationwide outbreak of contaminated artificial tears. Both cases were detected through database review of genomes in the Enhanced Detection System for Hospital-Associated Transmission (EDS-HAT), a routine genome sequencing-based surveillance program. We generated a high-quality reference genome for the outbreak strain from one of the case isolates from our center and examined the mobile elements encoding bla VIM-80 and bla GES-9 carbapenemases. We then used publicly available P. aeruginosa genomes to explore the genetic relatedness and antimicrobial resistance genes of the outbreak strain.

Impact of ompk36 genotype and KPC subtype on the in vitro activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam against KPC-producing K. pneumoniae clinical isolates.

Objectives: The availability of new ß-lactam/ß-lactamase inhibitors ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam have redefined contemporary treatment of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections. We aimed to characterize and contrast the in vitro activity of these agents against genetically diverse KPC-Kp clinical isolates. Methods: We analysed genomes of 104 non-consecutive KPC-Kp isolates and compared the in vitro antibiotic activity by KPC subtype and ompK36 genotype. MICs were determined in triplicate by CLSI methods. Twenty representative isolates were selected for time-kill analyses against physiological steady-state and trough concentrations, as well as 4× MIC for each agent. Results: Fifty-eight percent and 42% of isolates harboured KPC-2 and KPC-3, respectively. OmpK36 mutations were more common among KPC-2- compared with KPC-3-producing Kp (P < 0.0001); mutations were classified as IS5 insertion, glycine-aspartic acid insertion at position 134 (GD duplication) and other mutations. Compared to isolates with WT ompK36, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam MICs were elevated for isolates with IS5 by 2-, 4- and 16-fold, respectively (P < 0.05 for each). Against isolates with GD duplication, imipenem/relebactam and meropenem/vaborbactam MICs were increased, but ceftazidime/avibactam MICs were not. In time-kill studies, ceftazidime/avibactam-mediated killing correlated with ceftazidime/avibactam MICs, and did not vary across ompK36 genotypes. Imipenem/relebactam was not bactericidal against any isolate at trough concentrations. At steady-state imipenem/relebactam concentrations, regrowth occurred more commonly for isolates with IS5 mutations. Log-kills were lower in the presence of meropenem/vaborbactam for isolates with GD duplication compared with IS5 mutations. Conclusions: Our investigation identified key genotypes that attenuate, to varying degrees, the in vitro activity for each of the new ß-lactam/ß-lactamase inhibitors. Additional studies are needed to translate the importance of these observations into clinical practice.

Genomic Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Among University Students in Western Pennsylvania.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control on college campuses is challenging given communal living and student social dynamics. Understanding SARS-CoV-2 transmission among college students is important for the development of optimal control strategies. METHODS: SARS-CoV-2 nasal swab samples were collected from University of Pittsburgh students for symptomatic testing and asymptomatic surveillance from August 2020 through April 2021 from 3 campuses. Whole-genome sequencing (WGS) was performed on 308 samples, and contact tracing information collected from students was used to identify transmission clusters. RESULTS: We identified 31 Pangolin lineages of SARS-CoV-2, the majority belonging to B.1.1.7 (Alpha) and B.1.2 lineages. Contact tracing identified 142 students (46%) clustering with each other; WGS identified 53 putative transmission clusters involving 216 students (70%). WGS identified transmissions that were missed by contact tracing. However, 84 cases (27%) could not be linked by either WGS or contact tracing. Clusters were most frequently linked to students residing in the same dormitory, off-campus roommates, friends, or athletic activities. CONCLUSIONS: The majority of SARS-CoV-2-positive samples clustered by WGS, indicating significant transmission across campuses. The combination of WGS and contact tracing maximized the identification of SARS-CoV-2 transmission on campus. WGS can be used as a strategy to mitigate, and further prevent transmission among students.

Evolution of extended-spectrum ß-lactamase-producing ST131 Escherichia coli at a single hospital over 15 years.

Escherichia coli belonging to sequence type ST131 constitute a globally distributed pandemic lineage that causes multidrug-resistant extra-intestinal infections. ST131 E. coli frequently produce extended-spectrum ß-lactamases (ESBLs), which confer resistance to many ß-lactam antibiotics and make infections difficult to treat. We sequenced the genomes of 154 ESBL-producing E. coli clinical isolates belonging to the ST131 lineage from patients at the University of Pittsburgh Medical Center (UPMC) between 2004 and 2018. Isolates belonged to the well described ST131 clades A (8%), B (3%), C1 (33%), and C2 (54%). An additional four isolates belonged to another distinct subclade within clade C and encoded genomic characteristics that have not been previously described. Time-dated phylogenetic analysis estimated that the most recent common ancestor (MRCA) for all clade C isolates from UPMC emerged around 1989, consistent with previous studies. We identified multiple genes potentially under selection in clade C, including the cell wall assembly gene ftsI, the LPS biosynthesis gene arnC, and the yersiniabactin uptake receptor fyuA. Diverse ESBL genes belonging to the blaCTX-M, blaSHV, and blaTEM families were identified; these genes were found at varying numbers of loci and in variable numbers of copies across isolates. Analysis of ESBL flanking regions revealed diverse mobile elements that varied by ESBL type. Overall, our findings show that ST131 subclades C1 and C2 dominated and were stably maintained among patients in the same hospital and uncover possible signals of ongoing adaptation within the clade C ST131 lineage.

Genomic characterization of SARS-CoV-2 from vaccine breakthrough cases in Allegheny County, Pennsylvania.

We performed whole genome sequencing on SARS-CoV-2 from 59 vaccinated individuals from southwest Pennsylvania who tested positive between February and September, 2021. A comparison of mutations among vaccine breakthrough cases to a time-matched control group identified potential adaptive responses of SARS-CoV-2 to vaccination.

Predictive model for severe COVID-19 using SARS-CoV-2 whole-genome sequencing and electronic health record data, March 2020-May 2021.

OBJECTIVE: We used SARS-CoV-2 whole-genome sequencing (WGS) and electronic health record (EHR) data to investigate the associations between viral genomes and clinical characteristics and severe outcomes among hospitalized COVID-19 patients. METHODS: We conducted a case-control study of severe COVID-19 infection among patients hospitalized at a large academic referral hospital between March 2020 and May 2021. SARS-CoV-2 WGS was performed, and demographic and clinical characteristics were obtained from the EHR. Severe COVID-19 (case patients) was defined as having one or more of the following: requirement for supplemental oxygen, mechanical ventilation, or death during hospital admission. Controls were hospitalized patients diagnosed with COVID-19 who did not meet the criteria for severe infection. We constructed predictive models incorporating clinical and demographic variables as well as WGS data including lineage, clade, and SARS-CoV-2 SNP/GWAS data for severe COVID-19 using multiple logistic regression. RESULTS: Of 1,802 hospitalized SARS-CoV-2-positive patients, we performed WGS on samples collected from 590 patients, of whom 396 were case patients and 194 were controls. Age (p = 0.001), BMI (p = 0.032), test positive time period (p = 0.001), Charlson comorbidity index (p = 0.001), history of chronic heart failure (p = 0.003), atrial fibrillation (p = 0.002), or diabetes (p = 0.007) were significantly associated with case-control status. SARS-CoV-2 WGS data did not appreciably change the results of the above risk factor analysis, though infection with clade 20A was associated with a higher risk of severe disease, after adjusting for confounder variables (p = 0.024, OR = 3.25; 95%CI: 1.31-8.06). CONCLUSIONS: Among people hospitalized with COVID-19, older age, higher BMI, earlier test positive period, history of chronic heart failure, atrial fibrillation, or diabetes, and infection with clade 20A SARS-CoV-2 strains can predict severe COVID-19.

Genomic Diversity of Hospital-Acquired Infections Revealed through Prospective Whole-Genome Sequencing-Based Surveillance.

Healthcare-associated infections (HAIs) cause mortality, morbidity, and waste of health care resources. HAIs are also an important driver of antimicrobial resistance, which is increasing around the world. Beginning in November 2016, we instituted an initiative to detect outbreaks of HAIs using prospective whole-genome sequencing-based surveillance of bacterial pathogens collected from hospitalized patients. Here, we describe the diversity of bacteria sampled from hospitalized patients at a single center, as revealed through systematic analysis of bacterial isolate genomes. We sequenced the genomes of 3,004 bacterial isolates from hospitalized patients collected over a 25-month period. We identified bacteria belonging to 97 distinct species, which were distributed among 14 groups of related species. Within these groups, isolates could be distinguished from one another by both average nucleotide identity (ANI) and principal-component analysis of accessory genes (PCA-A). Core genome genetic distances and rates of evolution varied among species, which has practical implications for defining shared ancestry during outbreaks and for our broader understanding of the origins of bacterial strains and species. Finally, antimicrobial resistance genes and putative mobile genetic elements were frequently observed, and our systematic analysis revealed patterns of occurrence across the different species sampled from our hospital. Overall, this study shows how understanding the population structure of diverse pathogens circulating in a single health care setting can improve the discriminatory power of genomic epidemiology studies and can help define the processes leading to strain and species differentiation. IMPORTANCE Hospitalized patients are at increased risk of becoming infected with antibiotic-resistant organisms. We used whole-genome sequencing to survey and compare over 3,000 clinical bacterial isolates collected from hospitalized patients at a large medical center over a 2-year period. We identified nearly 100 different bacterial species, which we divided into 14 different groups of related species. When we examined how genetic relatedness differed between species, we found that different species were likely evolving at different rates within our hospital. This is significant because the identification of bacterial outbreaks in the hospital currently relies on genetic similarity cutoffs, which are often applied uniformly across organisms. Finally, we found that antibiotic resistance genes and mobile genetic elements were abundant and were shared among the bacterial isolates we sampled. Overall, this study provides an in-depth view of the genomic diversity and evolutionary processes of bacteria sampled from hospitalized patients, as well as genetic similarity estimates that can inform hospital outbreak detection and prevention efforts.

Carbapenem-Resistant Acinetobacter baumannii in U.S. Hospitals: Diversification of Circulating Lineages and Antimicrobial Resistance.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a major cause of health care-associated infections. CRAb is typically multidrug resistant, and infection is difficult to treat. Despite the urgent threat that CRAb poses, few systematic studies of CRAb clinical and molecular epidemiology have been conducted. The Study Network of Acinetobacter as a Carbapenem-Resistant Pathogen (SNAP) is designed to investigate the clinical characteristics and contemporary population structure of CRAb circulating in U.S. hospital systems using whole-genome sequencing (WGS). Analysis of the initial 120 SNAP patients from four U.S. centers revealed that CRAb remains a significant threat to hospitalized patients, affecting the most vulnerable patients and resulting in 24% all-cause 30-day mortality. The majority of currently circulating isolates belonged to ST2Pas, a part of clonal complex 2 (CC2), which is the dominant drug-resistant lineage in the United States and Europe. We identified three distinct sublineages within CC2, which differed in their antibiotic resistance phenotypes and geographic distribution. Most concerning, colistin resistance (38%) and cefiderocol resistance (10%) were common within CC2 sublineage C (CC2C), where the majority of isolates belonged to ST2Pas/ST281Ox. Additionally, we identified ST499Pas as the most common non-CC2 lineage in our study. Our findings suggest a shift within the CRAb population in the United States during the past 10 years and emphasize the importance of real-time surveillance and molecular epidemiology in studying CRAb dissemination and clinical impact. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAb) constitutes a major threat to public health. To elucidate the molecular and clinical epidemiology of CRAb in the United States, clinical CRAb isolates were collected along with data on patient characteristics and outcomes, and bacterial isolates underwent whole-genome sequencing and antibiotic susceptibility phenotyping. Key findings included emergence of new sublineages within the globally predominant clonal complex 2 (CC2), increased colistin and cefiderocol resistance within one of the CC2 sublineages, and emergence of ST499Pas as the dominant non-CC2 CRAb lineage in U.S. hospitals.