Identification of an Outbreak Cluster of Extensively Antibiotic-Resistant GC1 Acinetobacter baumannii Isolates in U.S. Military Treatment Facilities

ABSTRACT An outbreak involving an extensively antibiotic-resistant Acinetobacter baumannii strain in three military treatment facilities was identified. Fifty-nine isolates recovered from 30 patients over a 4-year period were found among a large collection of isolates using core genome multilocus sequence typing (MLST). They differed by only 0 to 18 single nucleotide polymorphisms (SNPs) and carried the same resistance determinants except that the aphA6 gene was missing in 25 isolates. They represent a novel sublineage of GC1 lineage 1 that likely originated in Afghanistan. IMPORTANCE A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts. Understanding how this organism enters and establishes itself within the hospital environment is key to interrupting transmission, but few genomic studies have examined these transmissions over a prolonged period. Though historical, this report provides an in-depth analysis of nosocomial transmission of this organism across continents and within and between different hospitals.

IMPORTANCE A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts. Understanding how this organism enters and establishes itself within the hospital environment is key to interrupting transmission, but few genomic studies have examined these transmissions over a prolonged period. Though historical, this report provides an in-depth analysis of nosocomial transmission of this organism across continents and within and between different hospitals. KEYWORDS Acinetobacter baumannii, GC1 lineage 1, outbreak identification, core genome MLST, epidemiology, microbial genomics, AMR, outbreak, antibiotic resistance, cgMLST, molecular epidemiology T he availability of modestly priced genome sequence data coupled with the development of varied bioinformatic tools has facilitated the identification of related and very closely related bacterial isolates (1). This helps improve infection control by allowing cases of transmission of a specific strain to be distinguished from polyclonal outbreaks. Here, we applied this approach to historical data to identify and track the spread of a specific sublineage of lineage 1 of the Acinetobacter baumannii global clone GC1 in and between military treatment facilities (MTF) from a likely origin in Afghanistan.
An example of this sublineage was previously reported during the emergence of tobramycin resistance following treatment of an extensively resistant infection in a U.S. service member injured in Afghanistan in 2010 (2). The initial isolate, MRSN 56, was susceptible only to the aminoglycosides amikacin and tobramycin and to tigecycline and colistin, and it developed tobramycin resistance during treatment via gene amplification (2,3). Recently, we reported the complete sequence of the MRSN 56 genome and identified the causes of the extensive antibiotic resistance in this strain (4).
Editor Daria Van Tyne, University of Pittsburgh School of Medicine This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
Using a cluster distance threshold of 10 alleles, MRSN 56 was determined to be part of a cgMLST cluster with 58 additional isolates cultured from 30 patients between 2007 and 2010 (Fig. 1). The average patient age was 29, all but two patients were male, and 29 patients had suffered traumatic blast injuries in Iraq (25.8%) or Afghanistan (71%), followed by medical evacuation to Germany (facility 2) and ultimately to MTFs in the United States (facilities 1 and 3). The 58 additional A. baumannii isolates exhibited the Outbreak of Acinetobacter baumannii from Afghanistan Microbiology Spectrum same resistance profile as MRSN 56, except that 33 were also resistant to amikacin (see below). Forty-four isolates were assigned to ST1 IP :KL1:OCL1, and the remaining 15 were assigned to ST1090 IP :KL1:OCL1, with ST1090 being a one-locus variant of ST1 (fusA1 to fusA151). A single nucleotide polymorphism (SNP)-based distance matrix of the 59 isolates was constructed using Snippy (https://github.com/tseemann/snippy) and revealed that they were separated by 0 to 18 SNPs (Fig. 1). Hence, this GC1 sublineage had spread within and between the three MTFs.
Unravelling the origins of this strain is complicated by the piecemeal collection and storage of isolates prior to the inception of the MRSN in 2009 (7), with many isolates not cryopreserved during this period. Though the earliest isolates in our repository were cultured from patients injured in Iraq, epidemiological data suggest that the original source of this strain was Afghanistan, as only patients injured in Afghanistan had A. baumannii cultured from groin surveillance swabs, indicating that they were colonized at the time of injury (Fig. 1). In support of an Afghan origin, two additional isolates, A. baumannii MRSN 571146 and MRSN 576822, were cultured from groin surveillance swabs of two Afghan nationals upon arrival at a U.S. military facility in Afghanistan (facility 4) in May 2018. Both isolates shared high genetic relatedness with MRSN 56 in the cgMLST analysis and were separated from the earlier isolates by 13 to 21 alleles (32 to 47 SNPs) (Fig. 1), providing evidence that a closely related strain is circulating in Afghanistan. Notably, both 2018 isolates are sequence type 1 (ST1) but carry K17 at the K locus in place of KL1. In addition to the same complement of antibiotic resistance genes in MRSN 56, the 2018 isolates also carried 3 additional antibiotic resistance genes; the carbapenemase gene bla NDM-1 , the aminoglycoside O-phosphotransferase gene aphA6 [aph(39)-VIa], conferring resistance to amikacin, and the strA [aph(30)-Ib] streptomycin resistance gene.
The cultivation of this A. baumannii strain from patient 24, who had no direct link to Afghanistan, highlights the likelihood of nosocomial transmission in and between facilities 1, 2, and 3 (Fig. 1). Patient 24, who was transferred directly to facility 1 from a country in Europe following a medical emergency in October 2009, had A. baumannii MRSN 2338 cultured 17 days after arrival despite having no direct connection to Iraq, Afghanistan, or facility 2. This isolate was essentially identical (separated by 1 SNP) to MRSN 2824, cultured from a surveillance swab of patient 23, who was injured in Afghanistan in September 2009 and was convalescing in the same room as patient 24 during this time. Similarly, all patients (except patient 24) were stabilized at facility 2 before transfer to facility 1 or 3, and there is strong evidence for nosocomial transmission there. For example, patient 15 was admitted to facility 2 in December 2008, and A. baumannii MRSN 32940 was cultured 3 days later. The patient was hospitalized for 21 days, during which time patient 16 arrived, and patient 16 grew A. baumannii MRSN 32941, which genetically identical to MRSN 32940 (0 SNPs), 14 days later.
The presence of aphA6 exactly correlated with amikacin resistance in the 35 resistant isolates (Fig. 1). To identify the context of aphA6, the draft genome (GenBank Outbreak of Acinetobacter baumannii from Afghanistan Microbiology Spectrum accession no. VHDR00000000) of amikacin-resistant MRSN 960 was reexamined.
Notably, in the contigs in the draft genome, the insertion sequence (IS) remnants at their boundaries that establish IS locations were removed by the Newbler assembler for low quality. Hence, the draft genome was reassembled (GenBank accession no. JAJEMU000000000) using Unicycler version 0.4.0, and the aphA6 gene was found to be in TnaphA6 (8) in the chromosome at the position corresponding to one of the copies of ISAba125 in MRSN 56 (at 7 o'clock in Fig. 1A in reference 4). The existence of only an ISAba125 at this position in MRSN 56 indicates that aphA6, which is flanked by directly oriented copies of ISAba125 in TnaphA6, has been lost from MRSN 56, probably via homologous recombination (Fig. 2).
In conclusion, access to large genomic data sets is providing heretofore-unparalleled insights into bacterial epidemiology and outbreak dynamics. In this study, we harnessed the data from over 3,000 A. baumannii genomes and uncovered a previously unrecognized multiyear outbreak of a new sublineage of GC1 lineage 1 across the military health system. A comparison of historical and contemporary isolates indicates that the strain likely originated in Afghanistan, where it continues to circulate and has since acquired the metallo-b-lactamase encoded by bla NDM-1 .
Data availability. The draft genomes of all 61 genomes in this study have been deposited in the NCBI GenBank database under project number PRJNA761133.

ACKNOWLEDGMENTS
The manuscript was reviewed by the Walter Reed Army Institute of Research and there is no objection to its presentation. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.