A de novo variant in PAK2 detected in an individual with Knobloch type 2 syndrome.

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.

Escherichia coli ST131 Associated with Increased Mortality in Bloodstream Infections from Urinary Tract Source.

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug-resistant clone, although its clinical impact on patients with bloodstream infection (BSI) is incompletely understood. This study aims to further define the risk factors, clinical outcomes, and bacterial genetics associated with ST131 BSI. A prospectively enrolled cohort study of adult inpatients with E. coli BSI was conducted from 2002 to 2015. Whole-genome sequencing was performed with the E. coli isolates. Of the 227 patients with E. coli BSI in this study, 88 (39%) were infected with ST131. Patients with E. coli ST131 BSI and those with non-ST131 BSI did not differ with respect to in-hospital mortality (17/82 [20%] versus 26/145 [18%]; P = 0.73). However, in patients with BSI from a urinary tract source, ST131 was associated with a numerically higher in-hospital mortality than patients with non-ST131 BSI (8/42 [19%] versus 4/63 [6%]; P = 0.06) and increased mortality in an adjusted analysis (odds ratio of 5.85; 95% confidence interval of 1.44 to 29.49; P = 0.02). Genomic analyses showed that ST131 isolates primarily had an H4:O25 serotype, had a higher number of prophages, and were associated with 11 flexible genomic islands as well as virulence genes involved in adhesion (papA, kpsM, yfcV, and iha), iron acquisition (iucC and iutA), and toxin production (usp and sat). In patients with E. coli BSI from a urinary tract source, ST131 was associated with increased mortality in an adjusted analysis and contained a distinct repertoire of genes influencing pathogenesis. These genes could contribute to the higher mortality observed in patients with ST131 BSI.

A multiplexed, three-dimensional pooling and next-generation sequencing strategy for creating barcoded mutant arrays: construction of a Schizosaccharomyces pombe transposon insertion library.

Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 variants Alpha and Iota.

SARS-CoV-2 variants shaped the second year of the COVID-19 pandemic and the discourse around effective control measures. Evaluating the threat posed by a new variant is essential for adapting response efforts when community transmission is detected. In this study, we compare the dynamics of two variants, Alpha and Iota, by integrating genomic surveillance data to estimate the effective reproduction number (Rt) of the variants. We use Connecticut, United States, in which Alpha and Iota co-circulated in 2021. We find that the Rt of these variants were up to 50% larger than that of other variants. We then use phylogeography to show that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of Alpha were larger than those resulting from Iota introductions. By monitoring the dynamics of individual variants throughout our study period, we demonstrate the importance of routine surveillance in the response to COVID-19.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The SARS-CoV-2 Delta variant rose to dominance in mid-2021, likely propelled by an estimated 40%-80% increased transmissibility over Alpha. To investigate if this ostensible difference in transmissibility is uniform across populations, we partner with public health programs from all six states in New England in the United States. We compare logistic growth rates during each variant's respective emergence period, finding that Delta emerged 1.37-2.63 times faster than Alpha (range across states). We compute variant-specific effective reproductive numbers, estimating that Delta is 63%-167% more transmissible than Alpha (range across states). Finally, we estimate that Delta infections generate on average 6.2 (95% CI 3.1-10.9) times more viral RNA copies per milliliter than Alpha infections during their respective emergence. Overall, our evidence suggests that Delta's enhanced transmissibility can be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on underlying population attributes and sequencing data availability.

Genomic heterogeneity underlies multidrug resistance in Pseudomonas aeruginosa: A population-level analysis beyond susceptibility testing.

BACKGROUND: Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. METHODS: Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. RESULTS: The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. CONCLUSIONS: The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta's infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta's enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 lineages.

Emerging SARS-CoV-2 variants have shaped the second year of the COVID-19 pandemic and the public health discourse around effective control measures. Evaluating the public health threat posed by a new variant is essential for appropriately adapting response efforts when community transmission is detected. However, this assessment requires that a true comparison can be made between the new variant and its predecessors because factors other than the virus genotype may influence spread and transmission. In this study, we develop a framework that integrates genomic surveillance data to estimate the relative effective reproduction number (R t ) of co-circulating lineages. We use Connecticut, a state in the northeastern United States in which the SARS-CoV-2 variants B.1.1.7 and B.1.526 co-circulated in early 2021, as a case study for implementing this framework. We find that the R t of B.1.1.7 was 6-10% larger than that of B.1.526 in Connecticut in the midst of a COVID-19 vaccination campaign. To assess the generalizability of this framework, we apply it to genomic surveillance data from New York City and observe the same trend. Finally, we use discrete phylogeography to demonstrate that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of B.1.1.7 were larger than those resulting from B.1.526 introductions. Our framework, which uses open-source methods requiring minimal computational resources, may be used to monitor near real-time variant dynamics in a myriad of settings.

Shared and distinctive features of the gut microbiome of C57BL/6 mice from different vendors and production sites, and in response to a new vivarium.

Animal models play a critical role in establishing causal relationships between gut microbiota and disease. The laboratory mouse is widely used to study the role of microbes in various disorders; however, differences between mouse vendors, genetic lineages and husbandry protocols have been shown to contribute to variation in phenotypes and to non-reproducibility of experimental results. We sought to understand how gut microbiome profiles of mice vary by vendor, vendor production facility and health status upon receipt into an academic facility and how they change over 12 weeks in the new environment. C57BL/6 mice were sourced from two different production sites for each of three different vendors. Mice were shipped to an academic research vivarium, and fresh-catch stool samples were collected from mice immediately from the shipping box upon receipt, and again after 2, 6 and 12 weeks in the new facility. Substantial variation in bacterial proportional abundance was observed among mice from each vendor at the time of receipt, but shared microbes accounted for most sequence reads. Vendor-specific microbes were generally of low abundance. Microbial profiles of mice from all vendors exhibited shifts over time, highlighting the importance of environmental conditions on microbial dynamics. Our results emphasize the need for continued efforts to account for sources of variation in animal models and understand how they contribute to experimental reproducibility.

Genome-Based Targeted Sequencing as a Reproducible Microbial Community Profiling Assay.

Current sequencing-based methods for profiling microbial communities rely on marker gene (e.g., 16S rRNA) or metagenome shotgun sequencing (mWGS) analysis. We present an approach based on a single-primer extension reaction using a highly multiplexed oligonucleotide probe pool. This approach, termed MA-GenTA (microbial abundances from genome tagged analysis), enables quantitative, straightforward, cost-effective microbiome profiling that combines desirable features of both 16S rRNA and mWGS strategies. The use of multiple probes per target genome and rigorous probe design criteria enabled robust determination of relative abundance. To test the utility of the MA-GenTA assay, probes were designed for 830 genome sequences representing bacteria present in mouse stool specimens. Comparison of the MA-GenTA data with mWGS data demonstrated excellent correlation down to 0.01% relative abundance and a similar number of organisms detected per sample. Despite the incompleteness of the reference database, nonmetric multidimensional scaling (NMDS) clustering based on the Bray-Curtis dissimilarity metric of sample groups was consistent between MA-GenTA, mWGS, and 16S rRNA data sets. MA-GenTA represents a potentially useful new method for microbiome community profiling based on reference genomes.IMPORTANCE New methods for profiling the microbial communities can create new approaches to understanding the composition and function of those communities. In this study, we combined bacterial genome-specific probe design with a highly multiplexed single primer extension reaction as a new method to profile microbial communities, using stool from various mouse strains as a test case. This method, termed MA-GenTA, was benchmarked against 16S rRNA gene sequencing and metagenome sequencing methods and delivered similar relative abundance and clustering data. Since the probes were generated from reference genomes, MA-GenTA was also able to provide functional pathway data for the stool microbiome in the assayed samples. The method is more informative than 16S rRNA analysis while being less costly than metagenome shotgun sequencing.

Emergence of Resistance to Ceftazidime-Avibactam in a Pseudomonas aeruginosa Isolate Producing Derepressed blaPDC in a Hollow-Fiber Infection Model.

Ceftazidime (CAZ)-avibactam (AVI) is a ß-lactam/ß-lactamase inhibitor combination with activity against type A and type C ß-lactamases. Resistance emergence has been seen, with multiple mechanisms accounting for the resistance. We performed four experiments in the dynamic hollow-fiber infection model, delineating the linkage between drug exposure and both the rate of bacterial kill and resistance emergence by all mechanisms. The Pseudomonas aeruginosa isolate had MICs of 1.0 mg/liter (CAZ) and 4 mg/liter (AVI). We demonstrated that the time at ≥4.0 mg/liter AVI was linked to the rate of bacterial kill. Linkage to resistance emergence/suppression was more complex. In one experiment in which CAZ and AVI administration was intermittent and continuous, respectively, and in which AVI was given in unitary steps from 1 to 8 mg/liter, AVI at up to 3 mg/liter allowed resistance emergence, whereas higher values did not. The threshold value was 3.72 mg/liter as a continuous infusion to counterselect resistance (AVI area under the concentration-time curve [AUC] of 89.3 mg · h/liter). The mechanism involved a 7-amino-acid deletion in the Ω-loop region of the Pseudomonas-derived cephalosporinase (PDC) ß-lactamase. Further experiments in which CAZ and AVI were both administered intermittently with regimens above and below the AUC of 89.3 mg · h/liter resulted in resistance in the lower-exposure groups. Deletion mutants were not identified. Finally, in an experiment in which paired exposures as both continuous and intermittent infusions were performed, the lower value of 25 mg · h/liter by both profiles allowed selection of deletion mutants. Of the five instances in which these mutants were recovered, four had a continuous-infusion profile. Both continuous-infusion administration and low AVI AUC exposures have a role in selection of this mutation.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Seroprevalence of SARS-CoV-2-Specific IgG Antibodies Among Adults Living in Connecticut: Post-Infection Prevalence (PIP) Study.

BACKGROUND: A seroprevalence study can estimate the percentage of people with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in the general population; however, most existing reports have used a convenience sample, which may bias their estimates. METHODS: We sought a representative sample of Connecticut residents, ages ≥18 years and residing in noncongregate settings, who completed a survey between June 4 and June 23, 2020, and underwent serology testing for SARS-CoV-2-specific immunoglobulin G (IgG) antibodies between June 10 and July 29, 2020. We also oversampled non-Hispanic black and Hispanic subpopulations. We estimated the seroprevalence of SARS-CoV-2-specific IgG antibodies and the prevalence of symptomatic illness and self-reported adherence to risk-mitigation behaviors among this population. RESULTS: Of the 567 respondents (mean age 50 [± 17] years; 53% women; 75% non-Hispanic white individuals) included at the state level, 23 respondents tested positive for SARS-CoV-2-specific antibodies, resulting in weighted seroprevalence of 4.0 (90% confidence interval [CI] 2.0-6.0). The weighted seroprevalence for the oversampled non-Hispanic black and Hispanic populations was 6.4% (90% CI 0.9-11.9) and 19.9% (90% CI 13.2-26.6), respectively. The majority of respondents at the state level reported following risk-mitigation behaviors: 73% avoided public places, 75% avoided gatherings of families or friends, and 97% wore a facemask, at least part of the time. CONCLUSIONS: These estimates indicate that the vast majority of people in Connecticut lack antibodies against SARS-CoV-2, and there is variation by race and ethnicity. There is a need for continued adherence to risk-mitigation behaviors among Connecticut residents to prevent resurgence of COVID-19 in this region.

CsrA Supports both Environmental Persistence and Host-Associated Growth of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic and frequently multidrug-resistant Gram-negative bacterial pathogen that primarily infects critically ill individuals. Indirect transmission from patient to patient in hospitals can drive infections, supported by this organism's abilities to persist on dry surfaces and rapidly colonize susceptible individuals. To investigate how A. baumannii survives on surfaces, we cultured A. baumannii in liquid media for several days and then analyzed isolates that lost the ability to survive drying. One of these isolates carried a mutation that affected the gene encoding the carbon storage regulator CsrA. As we began to examine the role of CsrA in A. baumannii, we observed that the growth of ΔcsrA mutant strains was inhibited in the presence of amino acids. The ΔcsrA mutant strains had a reduced ability to survive drying and to form biofilms but an improved ability to tolerate increased osmolarity compared with the wild type. We also examined the importance of CsrA for A. baumannii virulence. The ΔcsrA mutant strains had a greatly reduced ability to kill Galleria mellonella larvae, could not replicate in G. mellonella hemolymph, and also had a growth defect in human serum. Together, these results show that CsrA is essential for the growth of A. baumannii on host-derived substrates and is involved in desiccation tolerance, implying that CsrA controls key functions involved in the transmission of A. baumannii in hospitals.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates.

OBJECTIVES: To investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates. METHODS: Using a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target. RESULTS: Genomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/ß-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene. CONCLUSIONS: A novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

Distinct Mechanisms of Dissemination of NDM-1 Metallo-ß-Lactamase in Acinetobacter Species in Argentina.

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying blaNDM-1 Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. blaNDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying blaPER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.

A mutation in mouse Krüppel-like factor 15 alters the gut microbiome and response to obesogenic diet.

We identified a mouse strain, HLB444, carrying an N-ethyl-N-nitrosourea (ENU)-induced mutation in a highly conserved C2H2 zinc-finger DNA binding motif of the transcriptional regulator KLF15 that exhibits resistance to diet-induced obesity. Characterization of the HLB444 mutant model on high-fat and chow diets revealed a number of phenotypic differences compared to wild-type controls. When fed a high fat diet, HLB444 had lower body fat, resistance to hepatosteatosis, lower circulating glucose and improved insulin sensitivity compared to C57BL/6J controls. Gut microbial profiles in HLB444 generated from 16S rRNA sequencing of fecal samples differed from controls under both chow and high fat diets. HLB444 shares similar phenotypic traits with engineered full- and adipose-specific Klf15 knockout strains; however, some phenotypic differences between this mutant and the other models suggest that the Klf15 mutation in HLB444 is a hypomorphic variant. The HLB444 model will inform further annotation of transcriptional functions of KLF15, especially with respect to the role of the first zinc-finger domain.

Identification of Potential Virulence Factors in the Model Strain Acinetobacter baumannii A118.

Acinetobacter baumannii A118, a strain isolated from the blood of an infected patient, is naturally competent and unlike most clinical strains, is susceptible to a variety of different antibiotics including those usually used for selection in genetic manipulations. These characteristics make strain A118 a convenient model for genetic studies of A. baumannii. To identify potential virulence factors, its complete genome was analyzed and compared to other A. baumannii genomes. A. baumannii A118 includes gene clusters coding for the acinetobactin and baumannoferrin iron acquisition systems. Iron-regulated expression of the BauA outer membrane receptor for ferric-acinetobactin complexes was confirmed as well as the utilization of acinetobactin. A. baumannii A118 also possesses the feoABC genes, which code for the main bacterial ferrous uptake system. The functionality of baumannoferrin was suggested by the ability of A. baumannii A118 culture supernatants to cross feed an indicator BauA-deficient strain plated on iron-limiting media. A. baumannii A118 behaved as non-motile but included the csuA/BABCDE chaperone-usher pilus assembly operon and produced biofilms on polystyrene and glass surfaces. While a known capsular polysaccharide (K) locus was identified, the outer core polysaccharide (OC) locus, which belongs to group B, showed differences with available sequences. Our results show that despite being susceptible to most antibiotics, strain A118 conserves known virulence-related traits enhancing its value as model to study A. baumannii pathogenicity.

Rapid Replacement of Acinetobacter baumannii Strains Accompanied by Changes in Lipooligosaccharide Loci and Resistance Gene Repertoire.

The population structure of health care-associated pathogens reflects patterns of diversification, selection, and dispersal over time. Empirical data detailing the long-term population dynamics of nosocomial pathogens provide information about how pathogens adapt in the face of exposure to diverse antimicrobial agents and other host and environmental pressures and can inform infection control priorities. Extensive sequencing of clinical isolates from one hospital spanning a decade and a second hospital in the Cleveland, OH, metropolitan area over a 3-year time period provided high-resolution genomic analysis of the Acinetobacter baumannii metapopulation. Genomic analysis demonstrated an almost complete replacement of the predominant strain groups with a new, genetically distinct strain group during the study period. The new group, termed clade F, differs from other global clone 2 (GC2) strains of A. baumannii in several ways, including its antibiotic resistance and lipooligosaccharide biosynthesis genes. Clade F strains are part of a large phylogenetic group with broad geographic representation. Phylogenetic analysis of single-nucleotide variants in core genome regions showed that although the Cleveland strains are phylogenetically distinct from those isolated from other locations, extensive intermixing of strains from the two hospital systems was apparent, suggesting either substantial exchange of strains or a shared, but geographically restricted, external pool from which infectious isolates were drawn. These findings document the rapid evolution of A. baumannii strains in two hospitals, with replacement of the predominant clade by a new clade with altered lipooligosaccharide loci and resistance gene repertoires.IMPORTANCE Multidrug-resistant (MDR) A. baumannii is a difficult-to-treat health care-associated pathogen. Knowing the resistance genes present in isolates causing infection aids in empirical treatment selection. Furthermore, knowledge of the genetic background can assist in tracking patterns of transmission to limit the spread of infections in hospitals. The appearance of a new genetic background in A. baumannii strains with a different set of resistance genes and cell surface structures suggests that strong selective pressures exist, even in highly MDR pathogens. Because the new strains have levels of antimicrobial resistance similar to those of the strains that were displaced, we hypothesize that other features, including host colonization and infection, may confer additional selective advantages and contribute to their increased prevalence.