Examining the role of Acinetobacter baumannii Plasmid Types in Disseminating Antimicrobial Resistance

Acinetobacter baumannii is a Gram-negative pathogen responsible for hospital-acquired infections with high levels of antimicrobial resistance (AMR). The spread of multidrug-resistant A. baumannii strains, particularly those resistant to carbapenems, has become a global concern. Spread of AMR in A. baumannii is primarily mediated by the acquisition of AMR genes through mobile genetic elements, such as plasmids. Thus, a comprehensive understanding of the role of different plasmid types in disseminating AMR genes is essential. In this study, we analysed the distribution of plasmid types, sampling sources, geographic locations, and AMR genes carried on A. baumannii plasmids. A collection of 814 complete plasmid entries was collated and analysed. Most plasmids were identified in clinical isolates from East Asia, North America, South Asia, West Europe, and Australia. We previously devised an Acinetobacter Plasmid Typing (APT) scheme where rep/Rep types were defined using 95% nucleotide identity and updated the scheme in this study by adding 13 novel rep/Rep types (93 types total). The APT scheme now includes 178 Rep variants belonging to three families: R1, R3, and RP. R1-type plasmids were mainly associated with global clone 1 strains, while R3-type plasmids were highly diverse and carried a variety of AMR determinants including carbapenem, aminoglycoside and colistin resistance genes. Similarly, RP-type and rep-less plasmids were also identified as important carriers of aminoglycoside and carbapenem resistance genes. This study provides a comprehensive overview of the distribution and characteristics of A. baumannii plasmids, shedding light on their role in the dissemination of AMR genes. The updated APT scheme and novel findings enhance our understanding of the molecular epidemiology of A. baumannii and provide valuable insights for surveillance and control strategies. IMPORTANCE A. baumannii has emerged as a major cause of nosocomial infections, particularly in intensive care units, posing a substantial challenge to patient safety and healthcare systems. Plasmids, which carry antimicrobial resistance (AMR) genes, play a crucial role in the multidrug resistance exhibited by A. baumannii strains, necessitating a comprehensive understanding of plasmid spread, and how to track them. This study provides important insights into A. baumannii plasmid epidemiology, and the extent of their role in spreading clinically significant AMR genes and how they are differentially distributed across different clones i.e. sequence types (STs) and geographical regions. These insights are important for identifying high-risk areas or clones implicated in plasmid transmission, in the context of the spread of multidrug-resistant A. baumannii strains. It also highlights the involvement of R3-type, RP-type and rep-less plasmids in the acquisition and spread of significant AMR genes including those conferring resistance to carbapenems, aminoglycosides and colistin.


INTRODUCTION
the distribution of chromosomal sequence types, sampling, geographies, and AMR genes 75 carried on plasmids originally included in the APT database alongside an additional 193 76 complete plasmids that have since been deposited in GenBank (as of August 18, 2022). We also 77 provide an update to the original APT scheme with the addition of novel rep/Rep types. 78

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Overview of genome and plasmid dataset. As of August 18, 2022, 450 complete A. 80 baumannii genomes were available in GenBank. Of these 450 complete genomes, 80% (n=355) 81 had at least one plasmid (Table S1) with 236 genomes containing one (n=1) plasmid, and 113 82 carrying two plasmids (Table 1 and Table S1). Ninety-one (n=95) genomes lacked a plasmid 83 and were not studied here. To broaden our plasmid dataset, we extended our search to the 84 RefSeq database and captured an additional 92 genomes that contained at least one (n=1) 85 plasmids. Of these 92 genomes/unique strains, n=63 were not linked to a genome project and 86 n=29 genomes were sourced from WGS (Whole Genome Shotgun), which included draft 87 genomes with complete plasmid entries. Following curation of the dataset (i.e. exclusion of 88 duplicate entries and assembly QC; see methods for more details), our final dataset was 89 comprised of 814 non-redundant plasmid entries corresponding to at least 440 unique isolates 90 (Table S1 and Table S2; n=2/814 plasmids unassigned due to absence of BioSample and strain 91 sequences, we also report additional updates to the scheme as follows. R3-T49 has been 122 removed from the updated APT scheme, as the corresponding rep sequence (previously r3-123 T49_NZ_AYFZ01000080.1_pABUH2a-5.6_c33) has been identified as a R3-T26 variant. 124 Specifically, this variant carries an insert of 84 bp that differentiates this sequence from the 125 other R3-T26 variants. This entry has been subsequently renamed to R3-T26* and R3-T49 126 retired from the scheme. R1-T3 was also retired due to possible sequencing/assembly errors 127 resulting in shortening the Rep reading frame by approximately 150 amino acids. Lastly, we 128 highlight two corrections to Figure 3 and Table 3 published in the original APT paper 13 : i) R3-129 T3 was annotated twice in FIG 3; the first annotated clade highlighted in orange should be 130 corrected to R3-T8, and ii) in Table 3, the rows and columns should read R1-T1 to R1-T6 (i.e. 131 not P1-T1 to P1-T6). 132 Plasmids encoding the Rep_1 family replication protein (Rep_1 or R1 133 plasmids) do not carry AMR genes. R1-type plasmids (encoding Pfam01446) are often 134 2-3 kb in length and are typically comprised of a replication initiation protein and only two or 135 three additional open reading frames encoding hypothetical proteins. R1-type plasmids 136 constitute a small fraction of the plasmid dataset (n=16 plasmids, 13 isolates) and none of these 137 carried AMR genes, suggesting that these plasmids are not yet involved in the acquisition and 138 spread of AMR. Strains belonging to global clone 2 (GC2; largely represented by ST2) 139 constitute over 90% of all A. baumannii genomes in GenBank, but R1 plasmids appear to be 140 mainly associated with strains belonging to global clone 1 (GC1; largely represented by ST1, 141 n=9/13 isolates) with only n=1 GC2 strain found with R1 plasmids (FIG 2).
baumannii plasmid group. This is due to several reasons, including the Rep/rep sequence 145 divergence combined with their floating genetic structure arising from the presence of pdif 146 FIG 3). Over half of the plasmids were typed as R3 (n=479/814 147 plasmids; 59%), and these were detected in at least 345 unique isolates (note, n=2 R3 type 148 plasmids were unassigned to an isolate). Variants of R3-T1, T2 and T3 constitute the most 149 abundant types and were collectively detected in n=224 plasmids. R3-type plasmids appear to 150 be geographically dispersed, but some types appear to be limited to distinct regions (FIG 4) GenBank accession number CP031382.1), is illustrated in Figure S1. 194 In fact, RP-T1 and RP-T2 plasmids accounted for all AMR + RP-type plasmids. All n=49 RP-195 T1 AMR + plasmids carried either blaOXA-23 (carbapenemase; n=31 RP-T1 plasmids) and/or 196 aphA6 (amikacin resistance; n=30 RP-T1); n=12 RP-T1 plasmids carried both. Other AMR 197 genes detected in the RP-T1 plasmids included sul1, dfrA7,aacA4,strAB,aadA2,198 cmlA1, aadB, and the blaOXA-58 carbapenemase (FIG 5 and Table S2). Moreover, it appears that 199 variants of RP-T1 plasmids have similarly been acquired by all major globally distributed 200 clones including members of GC1 and GC2, ST10, ST15, ST25, ST79 and ST622, recovered 201 across all continents (FIG 5). 202 In contrast to the global distribution of RP-T1 plasmids, all n=22 RP-T2 plasmids were 203 sequenced from isolates collected in East Asia (predominantly China, except for one plasmid 204 with no AMR genes sequenced from an isolate in South Korea). Notably, n=15/22 plasmids 205 contained a blaOXA-23 copy suggesting that RP-T2 plasmids with blaOXA-23 are circulating in 206 China and have not yet been detected elsewhere. Plasmids corresponding to the remaining RP-207 types were generally small plasmids ranging in size from 4.5kb to 6.8kb (except for RP-T3; 208 52.5 kb) and carry no AMR genes. Interestingly, phylogenetic analysis of RP-type rep 209 sequences (RepPriCT_1 family) revealed a clear separation of the smaller plasmids that lack 210 AMR genes (RP-T4, RP-T5 and RP-T6) from the larger RP-T1, T2 and T3 plasmids ( Figure  211 X), suggesting distinct evolutionary trajectories that have likely influenced the accumulation 212 of additional genes including those conferring AMR. 213

Distribution of AMR genes in plasmids with no identifiable replication gene.
replication initiation gene (i.e. 22.9%; n=142/621 plasmids) 13 . Such plasmids might therefore 216 use an alternative mechanism that does not involve a Rep to initiate replication or encode a 217 novel Rep that is yet to be discovered. Here, n=161/814 plasmids did not encode an identifiable 218 replication initiation gene. This rep-less group constitutes a set of highly diverse plasmids 219 ranging in size from 4 kb to over 200 kb. Almost a third of these (n=52; 32.3%) appear to carry 220 no AMR genes and range in size from 2.4 -145.7 kb (Table 4). These plasmids are not 221 discussed further as they lack AMR genes. The remaining n=109 plasmids (length range 3.8 kb 222 to >200 kb) carry at least one AMR gene and constitute various plasmid variants. Some variants 223 are associated with the carriage of clinically significant AMR genes, and include those related 224 to pRAY*, large MPFF conjugative plasmids such as pA297-3, and pNDM-BK01 (n=28, 31 225 and 8, respectively; accounting for 41.6% rep-less plasmids). These plasmids are further 226 discussed below. 227 i) pRAY* -an important small plasmid spreading resistance to aminoglycosides. It has 228 been shown that the small plasmid pRAY* and its variants play a role in the spread of the aadB 229 gene conferring resistance to tobramycin, gentamicin and kanamycin, which are considered 230 clinically significant antimicrobials 25 . Although some variants did not carry an AMR gene (an 231 example shown in FIG 6), we observed n=28 plasmids that were either identical or closely 232 related to pRAY*, and most (n=25/28) carried aadB. These plasmids were found in strains 233 assigned to at least 14 STs, including ST1, ST81, ST2, ST25 and ST85 (Table S3). The strains ii) Spread of diverse AMR genes by conjugative plasmids encoding the MPFF transfer 239 system. This group constitutes a diverse set of n=31 large plasmids (146.7 -236.2 kb in size) 240 known to lack an identifiable rep gene. These plasmids were detected in at least 11 distinct STs 241 with the highest count corresponding to ST622 (n=10) followed by ST25 (n=7) and were also 242 present in members of the major global clones (e.g. ST1, ST10, ST25; Table S4). The 200.6 kb 243 plasmid, pA297-3 (Table S4) is considered the representative as it has the most common 244 backbone type and was one of the earliest described and shown to be conjugative 11 . It carries 245 sul2 and strAB, conferring resistance to sulphonamide and streptomycin respectively. Most 246 plasmids in this group (except p40288 and pR32_1; Table S4) carry a copy of sul2. Most 247 members also carry strAB (n=28/31), msr-mph(E) (n=21), blaPER-7 (n=18), and armA (n=20) 248 conferring resistance to streptomycin, macrolides, extended-spectrum β-lactamases (ESBLs), 249 and aminoglycosides, respectively (Table S4). The latter, armA, encodes the 16S rRNA 250 methylation protein that confers resistance to all aminoglycosides 26 . Two plasmids, 251 pPM193665_1 and pPM194122_1 (GenBank accession numbers CP050416 and CP050426, 252 respectively) from strains recovered in India, also contain the blaNDM metallo-ß-lactam 253 carbapenem resistance gene. 254 iii) Conjugative plasmids encoding MPFT transfer system. Though not very common, 255 blaNDM has now been reported in A. baumannii in several countries 3,27-30 . In this study, we 256 found n=8 plasmids with no identifiable rep gene that encode the MPFT type conjugative 257 transfer system 31 and carried the blaNDM metallo-beta-lactam carbapenem resistance gene 258 (Table 6). All these plasmids were found to be related to pNDM-BJ01 (GenBank accession 259 number JQ001791.1), which was first reported in Acinetobacter lowffii and shown to be 260 conjugative at a high frequency 32 . These plasmids were carried by strains recovered in clinical, US, Colombia, and Brazil showing their wide geographical distribution. They were found in 263 various sequence types, of which only one (p1AR_0088; GenBank accession number 264 CP027532.1; Table S2 and Table S5) was in a ST25 strain, which is an important globally 265 distributed ST 3,15,33-35 . Given the potential for accelerated resistance dissemination of 266 resistance to a last-line antimicrobial and hence heightened therapeutic challenges, targeted 267 surveillance of MPFT type plasmids with blaNDM may be warranted. 268 Diverse plasmid types facilitating the spread of carbapenem resistance genes.

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Carbapenemases stand out as important AMR determinants as carbapenems are one of the last 270 resort lines of defence in antimicrobial treatment 3 . Here, we showed that various R3, RP and 271 rep-less plasmids were associated with the spread of carbapenem resistance genes including 272 blaoXA-23, blaOXA-24, blaOXA-58, and blaNDM. Carbapenemases were observed in 150 plasmids 273 (18.4%), of which blaOXA-type genes were the most common carbapenemase type followed by 274 blaNDM (n=132, 11 and 7 plasmids with blaOXA only, blaNDM only and blaOXA plus blaNDM 275 respectively). We detected twelve allelic variants of blaOXA-type genes; blaOXA-23 was the most 276 prevalent (n=54), followed by blaOXA-58 (n=33, of which n=6 also carried blaNDM-1), blaOXA-72 277 (n=27), and blaOXA-24 (n=11). The prevalence of some of these alleles appear to be associated 278 with distinct plasmid types. For example, n=46/54 blaOXA-23 plasmids were typed as RP-T1 or 279 RP-T2, while R3-type plasmids appear to play a key role in the dissemination of blaOXA-58 (i.e. The blaNDM carbapenem resistance gene is clinically significant in all Gram-negative bacteria, 285 especially Enterobacterales as its rapid spread among different bacterial species worldwide has become a serious threat to public health 36 . A single allelic variant, blaNDM-1, was observed in 287 this dataset and detected in n=13 plasmids including n=8 pNDM-BJ01-type variants, n=2 R3-288 type n=2 pA297-3-type, and a novel plasmid pCCBH31258 (GenBank accession number 289 CP101888). The presence of blaNDM on conjugative plasmids in A. baumannii is significant as 290 it highlights the potential for the rapid transmission of this important carbapenemase via 291 horizontal gene transfer. 292 Opportunities and limitations. Advances in whole genome sequencing technologies 293 combined with the rapid accumulation of genome data in publicly available databases such as 294 GenBank has provided a valuable opportunity to gain genomic insights towards the circulation 295 of AMR genes in critical pathogens such as A. baumannii and, more importantly, MGEs that 296 disseminate AMR. However, this unique opportunity is associated with important caveats given 297 that publicly available genome sequences are largely geographically skewed for several 298 reasons, including the lack of technology, financial support and expertise in developing 299 countries. Currently, the bulk of genome sequence data in GenBank has been sequenced from 300 isolates collected in the US, China and Australia; these countries accounted for approx. ~50% 301 of the dataset in this study. The geographical skew of genome sequence data makes it difficult 302 to gain comprehensive insights into population structure, MGEs and AMR genes circulating in 303 other parts of the world e.g. Africa and the Middle East. Moreover, genomes of environmental 304 Traditionally, A. baumannii has been characterized as an organism that primarily acquires AMR 310 genes through large chromosomal islands. However, this definition is changing as more 311 plasmids that carry important AMR genes are being characterised. This study also highlights 312 the pivotal role of various plasmid types, particularly certain families in the dissemination of 313 clinically important AMR genes within this pathogen. We showed that many RP-T1, R3-types 314 (e.g. RP-T1 and R3-T2) and rep-less pNDM plasmids can spread various carbapenem 315 resistance genes. These are of particular concern as AMR genes conferring resistance to 316 carbapenems are often considered as the last line of defence in treatment. Furthermore, these 317 plasmids typically carry additional AMR genes conferring resistance to multiple 318 antimicrobials, which further compounds treatment management and the threat posed by A. 319

baumannii. 320
Although the plasmid repertoire of A. baumannii exhibits remarkable diversity, this 321 investigation highlights the profound significance of specific plasmid families in harboring and 322 disseminating AMR genes. The findings from this study provide new insights into which 323 plasmid types are over-represented among those that disseminate AMR and may be flagged as 324 targets for focused AMR surveillance. Finally, this study showed that, in the ongoing battle 325 against antibiotic resistance in A. baumannii, its plasmids play a significant role in exacerbating 326 the crisis. Their ability to transfer AMR genes across different sequence types, coupled with 327 the bacterium's adaptability, poses a formidable challenge to healthcare systems worldwide. 328 genome project' as data source in Table S1 and Table S2) and ii) an additional 92 335 genomes/unique strains (released between February 2021 and mid-August 2022) captured in 336

MATERIALS AND METHODS
RefSeq (https://www.ncbi.nlm.nih.gov/refseq/). The latter included n=29 genomes were 337 sourced from Whole Genome Shotgun projects (labelled as 'WGS' in Table SX) and n=63 338 unique strains that were not linked to a genome project (i.e. direct plasmid submission to 339 GenBank; labelled as 'GenBank non-redundant db' in Table S2). This resulted in the curation Bioinformatics and sequence analysis. The chromosomal sequences associated each plasmid 347 were found by exporting the BioSample accession numbers using the RefSeq 348 https://www.ncbi.nlm.nih.gov/refseq/ followed by the curation of a list of chromosomal 349