Epidemiological, Phylogenetic, and Resistance Heterogeneity Among Acinetobacter baumannii in a Large U.S. Deep South Healthcare system

Abstract Background Acinetobacter baumannii (Ab) disease in the United States is commonly attributed to outbreaks of 1 or 2 monophyletic carbapenem resistance (CR) Ab lineages that vary by region. However, there is limited knowledge regarding CRAb epidemiology and population structures in the U.S. Deep South, and few studies compare contemporary CR and carbapenem-susceptible (Cs) Ab, despite relative prevalence of the latter. Methods We performed a multiyear analysis of 2462 Ab cases in a large healthcare system in Birmingham, AL, and 89 post-2021 Ab isolates were sequenced and phenotyped by antibiotic susceptibility tests. Results Although the cumulative CR rate was 17.7% in our cohort, rates regularly increased in winter months as result of seasonal changes in case incidence of CsAb, specifically. Genotyped CRAb belonged to clonal group (CG) 1, CG2, CG108, CG250, or CG499, with local clones of CG108, CG250, and CG499 persisting over multiple months. There was no clonal expansion of any CsAb lineage. Among CRAb isolates, levels of β-lactam antibiotic resistance and the repertoire of related genetic resistance determinants, which included the novel CR-conferring FtsI A515V polymorphism, differed according to CG. CG108 and CG499 isolates displayed specific heteroresistance to sulbactam and trimethoprim/sulfamethoxazole, respectively, which resulted in discrepant susceptibility results in microbroth versus agar-based antibiotic susceptibility tests modalities. Conclusions We report an unusually high degree of CRAb phylogenetic diversity principally driven by emergent U.S. lineages harboring novel resistance elements that must be incorporated into diagnostic, surveillance, and preclinical research efforts.

Carbapenem-resistant (CR) Acinetobacter baumannii (Ab) is considered a top priority for research by the World Health Organization [1] and U.S. Centers for Disease Control and Prevention [2].This incidental pathogen is able to survive in various environments and regularly displays multidrug-resistant (MDR) phenotypes mediated by intrinsic Acinetobacter-derived cephalosporinase (ADC) class C β-lactamases and OXA class D β-lactamases, de novo polymorphisms in antibiotic targets, and a variety of extrinsic resistance genes-including OXA and non-OXA carbapenemases-acquired via mobile genetic elements (eg, plasmids, transposons).CRAb disease is conventionally linked to hospital-acquired (HA) respiratory, endovascular, wound, and urinary infections mainly attributable to a subset of MDR global lineages within the otherwise phylogenetically diverse species [3][4][5].
Ab strains are routinely genotyped according to 1 of 2 multilocus sequence type (MLST) schemes [6], and here we exclusively use the Pasteur MLST scheme [7].Single locus variant sequence types can be further grouped into monophyletic lineages, or clonal groups (CGs), often named according to the prevailing sequence type [8].CRAb-associated lineages CG2, CG1 and CG79 (the latter also known as Clade E [9]) comprised 61%, 5%, and 3%, respectively, of the ∼3500 genomes available on NCBI in early 2019 [10].CG2 comprised 85% of U.S. CRAb isolates in the first nationwide survey performed between 2008 and 2009 [11] and 64% of isolates from Centers for Disease Control and Prevention sentinel sites between 2013 and 2017 [12].In contrast, CG2 comprises a small portion of sequenced isolates from Latin American countries [8,10] and U.S. single-center studies reveal significant variability between regions and over time [9,[12][13][14][15].For example, most pre-2012 CRAb isolates in a St. Louis, Missouri, healthcare system were CG2 or CG79 Epidemiology of U.S Deep South A. baumannii • OFID • 1 Open Forum Infectious Diseases M A J O R A R T I C L E [11], but local clones of the unrelated ST406 (CG406, also known as USA-clone-1 [16]) and ST499 (CG499), the latter of which was first detected in Chicago in 2010 [17], rapidly became the prevailing CRAb lineages between 2017 and 2019 [14].
Awareness of Ab CGs propagating in a region could inform clinical practice because recent evidence suggests that CRAb lineages each display characteristic antibiotic susceptibility patterns and may differ in their epidemiological behavior [8,14].Notably, little is known regarding the population structure of Ab in the U.S. Deep South.Other than the inclusion of 6 Florida isolates [11] and a few Louisiana isolates reported during the writing of this manuscript [8], surveys have not included isolates from Deep South/Southeast region (including Tennessee, Mississippi, Georgia, and Alabama), where unique climate socioeconomic factors may selectively favor traits absent from pools of this environmental microbe in other regions.In addition, most recent surveys exclude cases associated with carbapenemsusceptible (Cs) Ab, despite these being a common cause of Ab disease and more representative of the heterogeneity within the species [14].
Here, we report findings from an ongoing investigation of Ab population dynamics in the U.S. Deep South.We compare the epidemiology of CsAb and CRAb isolates identified over 12 years in a large healthcare system in Birmingham, Alabama, and characterize the current population structure of clinically relevant Ab by isolate whole-genome sequencing.We report a link between emergent lineages and atypical antimicrobial resistance phenotypes and contextualize findings within the greater Ab population structure in the United States.

Study Location and Period
This study was approved by the University of Alabama at Birmingham (UAB) institutional review board (# 30003572 and 30008212) and was performed in the UAB Healthcare system from 1 July 2010 to 31 December 2022.UAB is the largest integrated inpatient and outpatient system serving Birmingham, Alabama, and surrounding areas.UAB Hospital and affiliated clinics use a central UAB Clinical Microbiology Laboratory (UAB-CML) on the main campus, which performed species identification of bacterial isolates by automated biochemical methods or matrix-assisted laser desorption/ionization and time-of-flight mass spectroscopy.Per routine protocol, the UAB-CML tests all Acinetobacter isolates via microbroth dilution antimicrobial susceptibility testing (MB-AST) using the Beckman Coulter Microscan WalkAway Plus AST system, against the following antibiotics: ampicillinsulbactam (SAM), cefepime (FEP), ceftazidime (CAZ), ciprofloxacin, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole (SXT), tobramycin, imipenem (IPM), and meropenem (MEM).
AST and susceptibility reporting is performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines [18].

Retrospective Study Design and Definitions
The UAB Cerner electronic medical record systems was queried using the Informatics for Integrating Biology and the Bedside tool hosted by the UAB Center for Clinical and Translation Science to identify cases and retrieve patient demographics, culture tissue source, hospital day of culture (if applicable), and AST interpretation results.Only first culture per patient ("index culture") identified over the course of routine care and containing the following was eligible for inclusion: "Acinetobacter baumannii" (n = 419), "Acinetobacter baumannii complex" (n = 651), "Acinetobacter baumannii complex/haemolyticus" (n = 5), or "Acinetobacter baumannii/haemolyticus" (n = 1387).Isolates reported as "intermediate" or "resistant" to either IPM or MEM were defined as "carbapenem nonsusceptible Ab (CNSAb).The CLSI Acinetobacter spp AST breakpoints for IPM and MEM were updated in 2014.Minimum inhibitory concentration (MIC) values were not retrievable in our retrospective cohort, so we relied on the carbapenem susceptibility interpretations according to breakpoints at the time of testing.A total of 169 of 186 (90.8%) pre-2015 CNSAb index isolates were reported "resistant" to at least 1 carbapenem, and, thus, meet CNSAb criteria using updated breakpoints.Cases were classified into 5 anatomical categories according to specimen source: "respiratory," "skin and soft tissue/musculoskeletal" (SST/MSK), "urinary," "blood" (including central lines, endovascular devices, or grafts), or "other."Cases were defined as HA if the index culture was performed ≥48 hours from time of hospitalization and before discharge.All other cases were defined as nHA.To evaluate seasonal trends, cases in 2011-2022 were grouped into 4 quarters according to the month index culture was obtained: 1 (January-March), 2 (April-June), 3 (July-September), and 4 (October-December).

Prospective Isolate Banking
Since November 2021, clinical isolates typed as an Acinetobacter species by matrix-assisted laser desorption/ionization and time-of-flight mass spectroscopy in the UAB-CML were identified via biweekly electronic medical record queries and were eligible for inclusion.For each colony morphotype identified as Acinetobacter on culture plates, 2-5 bacterial colonies were subcloned and transferred to the research laboratory for processing and storage.For this study, we arbitrarily chose 89 presumptive Ab isolates identified between 1 November 2021 and 31 November 2022 for sequencing and susceptibility testing analysis (Supplementary Data 1).Here, study isolates are named by number (ie, isolate 002 denotes UAB_Ab002, etc.).Ab isolates were not routinely banked at UAB before this study.

Whole-Genome Sequencing, Assembly and Comparative Genomics
A full description of the well-established processing pipelines [14] used for genome assembly and comparative genomics analyses is provided in the Supplementary Text.The earliest isolate with an MLST and/or antimicrobial resistance gene profile per person, was denoted as an index isolate and subsequent homologous isolates denoted as non-index isolates.

Kirby-Bauer AST
Study isolates were tested by Kirby-Bauer disk diffusion AST (KB-AST) in our research laboratory with Mueller-Hinton agar against SAM, FEP, CAZ, MEM, IPM, SXT, ciprofloxacin, gentamicin, and doxycycline.Zone of clearance breakpoints were determined and interpreted according to CLSI guidelines [18], and isolates "resistant" to MEM or IPM were designated "CRAb".In cases in which distinct colonies were repeatedly observed growing up to the border of the antibiotic disk but readily distinguishable from the lawn's major border, we subcloned bacteria from colonies at the disk border ("disk subclone") or from the main lawn ("lawn subclone").Subclones were plated and incubated at 37°C for 16 hours on LB Miller agar without antibiotics and retested by KB-AST the next day.Plate images were acquired from a height of 6.3 inches using an iPhone 14 Pro Max with 1× lens and arranged using Illustrator (Adobe, USA).

Global Analysis With Genomes From Other Contemporary U.S. Ab Isolates
Full description of the genome identification and comparative genomic meta-analysis is provided in the Supplementary Text.Briefly, an NCBI database query performed on 18 September 2023 identified 774 whole genome sequences of Ab isolated in the United States since 1 January 2010 per peer-reviewed reports.We performed de novo genome assembly using NCBI SRA files and, when available, extracted epidemiological and microbiological metadata (Supplementary Data 2).Genomes were binned into CGs according to MLST analysis, and comparative genomic analysis was done within each CG.In the case where a CG of interest was absent among these binned genomes (ie, CG108), we queried NCBI for homologous genomes whose metadata indicated they were from post-2009 U.S. isolates.

Statistical Analyses
Univariate analyses were performed with SPSS v29 (IBM, USA) or RStudio software v 4.1.2[19].Chi-squared or odds ratio test were performed for comparing categorical variables.Mann-Whitney test with Bonferroni adjustment for multiple comparisons was performed for continuous variables.Statistical significance was defined as P <.05.

Data Availability
All raw sequence files and assemblies derived from UAB study strains are available under NCBI BioProject PRJNA1005294 (see Supplementary Data 1).

Retrospective Analysis Reveals Diverse Clinical Presentations of Ab Cases at UAB
We identified 2462 cases associated with Ab index cultures between July 2010 and December 2022.A total of 478 cases (19.4%) contained CNSAb, of which 438 (17.7% of total Ab) tested "resistant" to MEM and/or IPM.The occurrence of CsAb, but not CNSAb, cases was higher in third calendar quarters (Figure 1A), and the resulting seasonal increase in Cs: CNS ratio led to an increased susceptibility rates among total Ab cases between May and October (Figure 1B).CNSAb cases were more likely than CsAb to be HA (54.2% [259/478] vs 41.6% [826/1984], odds ratio [95% confidence interval] = 1.66 [1.36-2.03]).However, HA rates among CNSAb decreased from 76.9% and 85.7% in late 2010 and early 2011, respectively, to 27.4% and 45.5% in both halves of 2022 (Supplementary Data 3).This trend coincided with later CNSAb cases being identified earlier during hospitalization, whereas CsAb HA rates and median hospital day of isolation were similar in all study periods (Figure 1C).CsAb and CNSAb displayed comparable distribution of isolate tissue sources when stratified according to onset (Figure 1D, top) (ie, respiratory isolates predominated among HA cases, while urinary and SST/MSK isolates predominated among nHA cases in nearly all study periods) (Figure 1D, bottom).Last, average patient age did not differ between CsAb (49.0 years) and CNSAb (50.3 years, P = .178)cases, but CNSAb was more likely to be isolated from males (odds ratio [95% confidence interval] = 1.49[1.21-1.84]).Females comprised less than 40% of CsAb respiratory cases (37.2%) and CNSAb respiratory (37.7%),SST/MSK (33.1%) urinary (34.7%), and other (31.6%)cases (Supplementary Table 1).In summary, CNSAb and CsAb were both implicated in various case types at UAB, albeit with differential features regarding seasonality, timing of index culture, and patient sex distribution.

2021-2022 UAB Ab Isolates Demonstrate Phylogenetic Heterogeneity
Genotyping of 89 Ab isolates identified in 2021-2022 revealed 42 STs (13 STs newly assigned in this study) distributed among 39 CGs (Supplementary Table 2).Ten case patients had multiple isolates included in our whole-genome sequencing analysis, but only 3 had 2 index isolates of distinct genotypes (Supplementary Figure 1).Thus, our cohort consisted of 73 index isolates from 68 individuals (Figure 2, Supplementary Data 1).A total of 58.9% (n = 43) of index isolates belonged to 9 CGs, each containing ≥2 index isolates obtained from independent Epidemiology of U.S Deep South A. baumannii • OFID • 3 cases occurring ≥30 days apart (Figure 2, Supplementary Table 2).Of these, CG108, CG164 and CG427 each contained isolates of 2 STs.The remaining index isolates (n = 30) each belonged to unrelated STs (Supplementary Data 4).
The identity of intrinsic OXA and ADC alleles was largely conserved within a lineage and rarely shared between lineages (Figure 3).Independent of OXA-23 presence, CG1 and CG2 isolates displayed greater FEP and CAZ resistance than other CsAb, including the CG250 CsAb isolates (Supplementary Figure 2).Conversely, CG250 and CG499 CRAb isolates displayed CAZ susceptibility indistinguishable from CG150 and other CsAb.CG108 isolates displayed the lowest detectable levels of susceptibility across all beta-lactam antibiotics in KB-AST (Supplementary Figure 2).We observed comparably greater intra-CC variation in the susceptibility to other antibiotic classes, which strongly correlated with the presence of resistance elements repeatedly found in multiple lineages (Figure 3 and Supplementary Figure 2).The only major discrepancy was SXT nonsusceptibility in CG499 isolates who all lacked the sul1/sul2 gene putatively conferring resistance in all other lineages, similar to CG499 isolates in prior studies [14].

Atypical Resistance Phenotypes Displayed by CG108 and CG499 Isolates
Results from clinical laboratory MB-AST generally matched KB-AST for most isolates, but 2 consistent discrepancies were noted (Supplementary Data 1, 5).First, CG108 isolates displaying the lowest detectable susceptibility to SAM in KB-AST were reported as "intermediate" or "susceptible" according to MB-AST.Second, despite CG499 isolates lacking canonical resistance genes and being "susceptible" to SXT via MB-AST, they displayed decreased susceptibility to SXT in KB-AST.Comparable MB-AST results were observed when banked isolates were retested in our research laboratory (Supplementary Data 1).Closer review of KB-AST plates found that CG108 isolates against IPM and SAM and CG499 isolates against SXT each displayed subpopulations extending between their main lawn margin and the antibiotic disk borders (Figure 4A).Bacteria from the lawn or colonies at the antibiotic disk border were subcloned overnight without antibiotics, and secondary KB-AST was performed (Figure 4B).Each subclone pair yielded similar results in secondary KB-AST (Figure 4C-D).Similar SXT-related phenomenon was observed when testing unrelated ST499 isolates from St. Louis (Supplementary Figure 3) [14].
No other lineages displayed similar patterns in KB-AST.

Comparative Analysis With Ab Isolates From Other U.S. Regions
The diversity of CRAb lineages among isolates was unexpected, so we investigated how the UAB cohort related to the greater U.S. Ab population.We identified 545 genomes from Ab isolated in the United States after January 1 2010 (Supplementary Data 2) for intra-CG comparative analyses (Supplementary Table 2).UAB CG2 isolates occupied 3 branches within 2 previously described clades [9,21], 1 branch in Clade C (isolates 021 and 187) and 2 in Clade A (isolate 028 and all the others, respectively) (Figure 5).Most CG1, CG499, CG108, and CG250 UAB isolates occupied exclusive branches within their lineages, though 2 CG250 (isolates 024 and 232) and CG108 isolates (isolates 076 and 260) were on distinct branches.Conversely, UAB CG150 and CG32 genomes were broadly distributed within their respective trees.No CG427 or CG164 isolates were identified among genomes from other regions, and 2 of the most common U.S. lineages, CG406 and CG79, were absent from the UAB cohort (Supplementary Table 2).
ARG analysis of the total cohort revealed intra-CG variability regarding strains harboring resistance genetic elements for non-β-lactam antibiotic classes (Supplementary Data 2).In contrast, the allelic identity of intrinsic OXA and ADC genes facilitated the delineation of phylogenetic subclades within CGs, most of which comprised isolates from different studies and regions (Supplementary Figure 4).Though OXA-23 was the most commonly encoded carbapenemase overall, OXA-24/72 carbapenemases were identified in CG2 subclades A2, C2, and G (Supplementary Figure 4B), half of CG79, 2 unrelated subclades in CG406, and nearly all non-Mountain region CG499 isolates (Supplementary Figure 4C).Notably, the FtsI A515 V polymorphism was encoded by isolates only in CG2 subclade B2 (Supplementary Figure 4B) and in CG108 subclades containing isolates lacking any carbapenemase (Supplementary Figure 4C).CG406 was the only CRAb-associated lineage [8,14] where most isolates lacked an identifiable carbapenem resistance element.No extrinsic β-lactamase genes were identified among CG150 and CG32 isolates (Supplementary Figure 4D).

DISCUSSION
We report a comprehensive analysis of clinical cases associated with Ab isolates in a major U.S. Deep South healthcare center.With the aim of delineating the microbial landscape of Ab propagating in our region, we characterized the presentation of Ab-associated cases and the population structure of clinical isolates.Investigation of clinical outcomes and transmission dynamics is the subject of ongoing study, especially because many of the lineages identified in the region were absent in recent major studies [8].
CsAb cases are more prevalent than CRAb in many U.S. regions [5,22], but are often excluded from epidemiological surveys.We found that HA rates among CRAb cases approximated those of CsAb cases in later years (Supplementary Data 3) and isolate tissue source distributions were indistinguishable (Figure 1E-F).However, consistent with reports from other regions [5,23], seasonality only occurred among CsAb cases (Figure 1A-B).The CsAb and CRAb population structures also differed: 37 unrelated lineages were identified among 47 CsAb isolates, but only 5 CGs were observed among 26 CRAb.Two CsAb lineages (ie, CG150 and CG32) were associated with multiple independent cases in our cohort.However, these CsAb isolates did not appear to share recent common ancestors (Figure 5), and CG150 or CG32 isolates were only spuriously identified in other U.S. studies (Supplementary Figure 4D).Repeated observations of decreased relatedness among CsAb isolates [14] supports that CsAb disease is result of sporadic crossover events from environmental sources.
In contrast, we identified multiple CRAb clonal clusters comprising isolates collected ≥90 days apart, from different tissue sources, and associated with HA and nHA cases (Figure 2).Though many of these isolates belong to subclades unique to our region, attributing CRAb clusters to hyperlocalized outbreaks would require a formal clonality analysis beyond the scope of our current study.Regardless, these observations imply the existence of contemporary non-hospital CRAb pools persisting in the region's patient population, as observed in other U.S. studies [9,14].
We observed an unexpectedly high degree of CRAb lineage heterogeneity of in our cohort.As an environmental species, Ab harbors a very diverse genetic pool even compared to other Gram-negative pathogens [24,25], but recent studies have repeatedly shown only 1 or 2 lineages predominating in each healthcare center [13,14].It can be speculated that climate and/or socioeconomic factors in the U.S. Deep South are more permissive to Ab phylogenetic diversity than other regions [8,12], akin to comparing Ab populations in Latin America to the rest of the world [8,10].Thus, we predict investigation of Ab in other Deep South regions will detect similar heterogeneity.
Historically, most CRAb disease has been attributed to CG2 [8,9,13,15], but new lineages have emerged across the United States in recent decades.CG499 was first described in 2008 (as a single ST123 isolate [11]) and 2010 (as a ST499 isolate [17]) but has now become the second most common CRAb lineage in the United States and the dominant lineage in some centers [8,12,14].CG108 was only sporadically observed in prior studies, but we demonstrate its capacity to establish a stable presence in a region.ST250 was first identified in a 2007 isolate from Puerto Rico [26], rarely identified in prior U.S. surveillance, but repeatedly isolated in our cohort.Last, though not identified in our cohort, CG406 has been exclusively identified in various U.S. regions in recent years [8,14,15].Altogether, generalizing historical or even multicenter study findings to a region's Ab population structures should be avoided because the lineages responsible for local cases can differ drastically from site to site and over time.
The contribution of Ab phylogeny to clinical outcomes is unclear [3,8].We argue, however, that surveilling local CRAb lineages' unique resistance features can influence local medical practices.For example, as the most prevalent carbapenemase among CRAb, OXA-23 is a target of current and emergent rapid molecular detection tools in clinical labs [27].However, U.S. isolates belonging to CG2, CG108, and CG499 display carbapenem resistance via other genetic determinants (ie, OXA-24 or the FtsI A515 V polymorphism), and the mechanism of resistance in many CG406 isolates remains to be identified [8,14,16] (Supplementary Figure 4).Indeed, OXA-23 was detected in <50% of 6026 CRAb isolates tested in the Antimicrobial Resistance Laboratory Network [28].Last, as previously reported [14], we observed that the level of resistance conferred by a β-lactamase varies from lineage to lineage (ie, OXA-23-associated IPM resistance was greater among CG2 isolates compared to CG250 isolates) (Supplementary Figure 2).Altogether, the utility of OXA-23-based testing varies according to regional CRAb population structures.
Another important finding was the discordance between MB-AST and KB-AST for SAM against CG108 isolates and for SXT against CG499 isolates.The repeated occurrence of subpopulations with different resistance levels and the instability of the increased resistance phenotype (Figure 4) are suggestive of phasevariable heteroresistance in these CGs.How these phenotypes are achieved is unclear, but the strict link between lineage and antibiotic class suggests involvement of separate mechanisms.This is especially true for CG499, which appears to achieve decrease susceptibility to SXT without any of the typical genetic elements associated with SXT resistance in other Ab lineages (Figure 3).Regardless, why these traits are not readily identifiable via MB-AST and how the spontaneous occurrence of increased SAM or SXT resistance influences clinical outcomes or susceptibility to new combination agents (ie, sulbactam-durlobactam) should be the focus of more immediate study.
Our work shows how knowledge of isolate lineage could influence clinical decision-making and sets a foundation to explore how lineage may influence other aspects of Ab disease, such as virulence, clinical prognosis, and propensity to be spread among certain patient populations.Though consistent with recent reports from other U.S. locations [12][13][14], our study was performed in a single healthcare center, and these early-stage findings encompass only a year's worth of isolates.Whether our findings are generalizable to nationwide trends or whether other centers in the U.S. Deep South harbor Ab populations with elevated degree of heterogeneity compared to other U.S. regions must be further investigated.

Figure 1 .
Figure 1.Divergent epidemiological features of Ab cases at UAB, July 2010-December 2022.A, Proportion of annual carbapenem-susceptible A. baumannii (CsAb) and carbapenem nonsusceptible Ab (CNSAb) cases occurring in each calendar quarter (Q1-Q4) during 2011-2022.**< .001.B, Cumulative case total for each month (left y-axis) of CsAb and CNSAb (light and dark gray, respectively) and monthly CNS rate (dotted line, right y-axis) among cases from 2011 to 2022.C, Semiannual totals (left y-axis) of hospital-acquired (HA) and non-hospital acquired (nHA) CNSAb (top) and CsAb (botom) cases.Lines and error bars depict the median and interquartile range (IQR) for hospital day of isolation among cases in each period (right y-axis).D, Cumulative (top) and semiannual (bottom) percentage of CNSAb and CsAb index isolates from each tissue source, separated by nHA (left) and HA (right) cases.

Figure 2 .
Figure 2. Diversity of Ab isolates at UAB hospitals, 2021-2022.Maximum likelihood phylogenetic tree of UAB Ab index isolates according to alignment of 2072 core genes, rooted to the genome of reference strains 19 606 (labeled).Inner label ring denotes CG with more >1 index isolate.Filled circles denote CRAb and isolates associated with HA cases.Outer rings represent isolate metadata according to the corresponding keys, as listed in Supplementary Data 1.

Figure 3 .
Figure 3. Antibiotic susceptibility and resistance genetic element content of UAB index isolates (top).Tested antibiotics are listed on left and grouped by antibiotic class.Results are presented as "resistant" (dark red), "intermediate" (light pink), or "susceptible" (white) according to CLSI guideline interpretation of Kirby-Bauer results.Circles below each AST group denotes presence (fill circle) or absence of associated resistance genetic elements listed on right.Intrinsic OXA and ADC alleles are listed by allele number.For clarity, only genetic elements present in isolates belonging to multi-isolate CGs (boxed columns with labels below) are included.Results for all UAB isolates and genetic elements are listed in Supplementary Data 4.

Figure 4 .
Figure 4. KB-AST identifies susceptibility-variable subpopulations associated with CRAb lineage and antibiotic type.A, Representative pictures of the most commonly observed KB-AST results for main CGs in our cohort (left), against different antibiotics (top).Red letters denote samples that were subcloned and incubated overnight on LB agar with no antibiotic followed by secondary KB-AST, as depicted in panel B. C-D) Secondary KB-AST of "lawn" (left) and "disk" (right) subclones.Photos show results from multiple antibiotics (top) and magnification of zones of clearance around antibiotics of interest (bottom).Abbreviations: CAZ, ceftazidime; FEP, cefepime; GM, gentamicin; IPM, imipenem; MEM, meropenem; SAM, ampicillin-sulbactam; SXT, trimethoprim/sulfamethoxazole.