Contributions of plasmid p1AB5075-encoded antibiotic resistance genes to multidrug resistance of Acinetobacter baumannii AB5075

13 Infections with m ultidrug resistant Acinetobacter baumannii are considered a threat to human and 14 animal health. The widely studied A. baumannii strain AB5075 displays a high degree of antibiotic 15 resistance. In this study, we experimentally validated that antibiotic resistance is largely mediated by 16 resistance genes located on plasmid p1AB5075. We used a p1AB5075-deficient AB5075 strain to 17 assess individual contributions of p1AB5075-encoded antibiotic resistance gene s by ectopically 18 (over-)expressing each gene in the Δp1AB5075 background. By determining individual contributions 19 of seven p1AB5075-encoded antibiotic resistance genes, we show individual and overlapping roles 20 of genes for aminoglycoside resistance and uncover the importance of extended-spectrum ß-21 lactamase blaGES-11 for cephalosporin resistance in A. baumannii AB5075. We discovered that 22 aminoglycoside N -acetyltransferase aaC(6’)-Ib3 (aacA4), which was considered a potential 23 pseudogene in A. baumannii AB5075, to be functional providing broad resistance to gentamicin, 24 kanamycin, amikacin, streptomycin and tobramycin when overexpressed in A. baumannii AB5075. 25 Because p1AB5075 is transferrable to a wide range of environmental and clinical A. baumannii 26 strains and non-baumannii Acinetobacter species, the relevance of our findings extends beyond 27 A. baumannii AB5075. 28 29 30 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 2

31 Curing infections with multidrug resistant (MDR) microorganisms of humans and animals is one of 32 the greatest medical challenges of the 21st century. Among the most problematic bacteria that require 33 investment into research and development of new antibiotics or alternative treatment strategies are 34 carbapenem-resistant Acinetobacter baumannii (Tacconelli et al., 2018; Whiteway et al., 2022a; Sati 35 et al., 2025). Infections with A. baumannii are increasingly difficult to clear because of a high degree 36 of antimicrobial resistance, strain heterogeneity and reports of pan-drug resistant strains (Valcek et 37 al., 2022a; Cain and Hamidian, 2023; Valcek et al. , 2025) . Understanding the evolution of 38 antimicrobial resistance and resistance mechanisms may inform the development of new drugs and 39 better treatment regimes. Early isolates that have been extensively used to study A. baumannii biology 40 were typically sensitive to antibiotics including the widely studied strains A. baumannii ATCC17978 41 and ATCC19606 (Smith et al. , 2007) . Contemporary A. baumannii strains, including the widely 42 studied A. baumannii AB5075, are likely more suitable representatives of current infections, and are 43 often resistant to a large array of antibiotics (Jacobs et al., 2014). In A. baumannii AB5075, many 44 antibiotic resistance genes (ARGs) are located on plasmid p1AB5075 within “Resistance Island 2” 45 (RI-2), but some also are found on the chromosome (e.g., oxa-23) (Jacobs et al., 2014; Gallagher et 46 al., 2015). The plasmid p1AB5075 is the largest of three plasmids in A. baumannii AB5075 (83,160 47 bp), which was shown to be transferrable to other A. baumannii strains of environmental and clinical 48 origin and to diverse non -baumannii Acinetobacter species (Jacobs et al. , 2014; Gallagher et al., 49 2015; Nasser et al. , 2024; Martz et al. , 2025) . The RI -2 locus is chiefly responsible for 50 aminoglycoside (hetero-)resistance in A. baumannii AB5075, which is caused in part by a RecA-51 dependent resistance island amplification to up to 20 -24 copies (Anderson et al., 2018). The RI-2 52 small RNA SrvS located upstream of aadB was also shown to be involved in virulent opaque (VIR-53 O) to avirulent translucent colony (A V-T) type switching, showing that p1AB5075-encoded genes are 54 engaging in regulatory cross-talk between p1AB5075 and the chromosome (Anderson et al., 2020). 55 In addition, another opaque subpopulation of AB5075 termed LSO (low switching opaque) is distinct 56 from the VIR -O variant by reduced switching frequency compared to VIR -O. The number of RI2 57 copies (the LSO variant has only one copy, while the VIR -O variant has ≥2 copies) influenc es 58 switching frequency between opaque and translucent variants through the copy number of the srvS 59 gene (Anderson et al., 2020). RI-2 is mosaic and contains among other ARGs an extended-spectrum 60 β-lactamase blaGES-11, which was first described in an A. baumannii strain isolated in France 61 (Moubareck et al., 2009). Despite their biological importance, s pontaneous loss of large plasmids, 62 including p1AB5075, has been documented for A. baumannii (Valcek et al., 2025). Plasmid pAB3 63 has been reported to have been lost in A. baumannii ATCC17978, which caused up-regulation of the 64 Type-6 Secretion System (Weber et al. , 2015; Kröger et al. , 2018) , and loss of p1AB5075 in 65 A. baumannii AB5075 resulted in sensitivity to amikacin and tobramycin and reduced resistance to 66 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 3 chloramphenicol (Anderson et al., 2020; de Dios et al., 2022). Here, we report loss of p1AB5075 in 67 A. baumannii AB5075 independent from the published reports (Anderson et al., 2020; de Dios et al., 68 2022). We used the Δp1AB5075 genetic background to characterise the antibiotic resistance profile 69 for the p1AB5075 -deficient strain and genetically dissect the contributions of seven individual 70 p1AB5075-encoded antibiotic resistance genes for high level multidrug resistance. 71 72

73 Bacterial strains and general growth conditions . Acinetobacter baumannii AB5075 and 74 Escherichia coli TOP10 (Invitrogen) were maintained on lysogeny broth (Lennox, L-) agar plates (10 75 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar) and grown over night in liquid L-broth, 10 g/l 76 tryptone, 5 g/l yeast extract, 5 g/l NaCl) (Jacobs et al., 2014). Media contained tetracycline (12 μg/ml) 77 when necessary to maintain plasmid pWH1266 (Hunger et al., 1990). 78 Strain and p lasmid construction s. ARGs were amplified from purified genomic DNA of 79 A. baumannii AB5075 using DNA oligonucleotides listed in Supplementary Table 1 with ~20 bp 80 overhangs complementary to the pWH1266 backbone to enable sequence and ligation independent 81 cloning extract (SLiCE)-mediated cloning (Zhang et al., 2012). Polymerase chain reaction (PCR) 82 amplification of DNA was performed using VerifyTM polymerase (PCR Biosystems). Plasmid 83 isolations and PCR purification s were carried out according to the manufacturer ’s protocol using 84 EasyPure Plasmid MiniPrep and PCR Purification Kits (TransGen Biotech ). SLiCE cloning 85 procedure was used to insert ARGs into the PCR-amplified pWH1266 backbone (Zhang et al., 2012). 86 All plasmid construction steps were carried out in E. coli TOP10 cells. To ensure efficient and equal 87 translation of ARG mRNAs, the forward primer contained an artificial AGGAGG ribosome binding 88 site (RBS). The cloned genes were constitutively expressed from the β-lactamase (bla) promoter of 89 pWH1266 (Hunger et al., 1990). Plasmids were re-isolated from E. coli, their insert sequence verified 90 by Sanger sequencing (Eurofins), and AB5075 was subsequently transformed with the plasmids using 91 natural transformation or electroporation (Godeux et al. , 2020) . Whole plasmid sequencing of 92 pWH1266 was conducted by Azenta/GENEWIZ (GENEWIZ Germany GmbH) using Oxford 93 Nanopore Technology (ONT) in a GridION with a FLO -MIN114 (R10.4.1) flow cell (GenBank 94 accession no.: PV577797.1). 95 Disk diffusion assays. All A. baumannii AB5075 strains were cultured overnight in cation-adjusted 96 Mueller-Hinton broth (MH2, Merck/Millipore). Overnight cultures were adjusted to 0.5 McFarland 97 standard, and each culture sample was swabbed thoroughly onto Mueller-Hinton agar plates using a 98 sterile cotton swab . Plates were air-dried for 10 min before addition of antimicrobial susceptibility 99 disks (O xoid). Antibiotic disks contained amikacin (AK, 30 μg), tobramycin ( TOB, 10 μg), 100 kanamycin ( K, 30 μg), gentamicin ( CN, 10 μg), trimethoprim -sulfamethoxazole ( SXT, 25 μg), 101 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 4 streptomycin (S, 25 μg), meropenem (MEM, 10 μg), doripenem (DOR, 10 μg), imipenem (IMP 10 102 μg), vancomycin (V A, 30 μg), erythromycin (E, 15 μg), nitrofurantoin (F, 100 μg), tetracycline (TE, 103 30 μg), tigecycline ( TGC 15 μg), ciprofloxacin (CIP, 5 μg), cefepime (FEP, 30 μg), cefoperazone 104 (CFP, 30 μg), ceftazidime (CAZ, 30 μg), chloramphenicol (C, 30 μg), aztreonam (ATM, 30 μg), 105 oxacillin (OX, 1 μg), ticarcillin (TIC, 75 μg), cefoxitin (FOX, 30 μg) and piperacillin/tazobactam 106 (TZP, 110 μg). Plates were incubated over night at 37°C before measuring the diameters of inhibition 107 zones. 108 Broth microdilution assay. Broth microdilution assays were performed as per the Clinical & 109 Laboratory Standards Institute (CLSI) guidelines for Acinetobacter to determine the Minimum 110 Inhibitory Concentrations (MIC) for antibiotics. The assays were performed in 96-well plates. Ten µl 111 of culture (0.5 McFarland) was added to each well except for the negative control (MH2 medium 112 without bacteria). Plates were incubated at 37 °C statically for 22 h before the plates were analysed 113 visually. 114

115 Loss of p1AB5075 results in extensive sensitivity to multiple antibiotics. 116 Acinetobacter baumannii AB5075 is considered a multidrug resistant strain with increased virulence 117 and a contemporary representative of A. baumannii infections (Jacobs et al. , 2014) . Because 118 A. baumannii, and especially A. baumannii AB5075, shows a high degree of phenotypic and 119 genotypic heterogeneity (Chin et al., 2018; Whiteway et al., 2022b; Pérez-Varela et al., 2022; Valcek 120 et al. , 2022b; Cooper et al. , 2024; Singh et al. , 2025; Valcek et al. , 2025) , we whole-genome 121 sequenced a number of A. baumannii AB5075 colonies after attempting to delete genes AB5075 by 122 the suicide-vector method of Pokhrel et al. (Pokhrel et al., 2023). Serendipitously, this revealed that 123 one of the sequenced colonies, where the wild-type genotype at the position of the attempted deletion 124 was restored, had lost plasmid p1AB5075 but was otherwise wild -type—an event which had been 125 independently reported before (de Dios et al. , 2022) . As p1AB5075 carries multiple antibiotic 126 resistance genes as part of R I-2 (Gallagher et al., 2015), we set out to characterise the antibiotic 127 resistance profile of the Δp1AB5075 strain and to investigate the individual contributions of ARGs 128 on p1AB5075, which so far, with the exception of cmlA contributing to chloramphenicol resistance 129 had not been experimentally verified gene-by-gene in A. baumannii AB5075 (de Dios et al., 2022). 130 The predicted ARGs of p1AB5075 are shown in the context of p1AB5075 in Figure 1 and their 131 annotation is presented in Table 1. All ARGs except one , a predicted APH(3')-VI family 132 aminoglycoside O-phosphotransferase (adh/ABUW_RS19420), are located within RI -2 (Figure 1A 133 & B). Antibiotic disk diffusion assays were performed to assess antibiotic resistance profiles of wild-134 type AB5075 and the Δp1AB5075 mutant strain to a suite of antibiotics . The Δp1AB5075 mutant 135 strain showed widespread increased susceptibility including to all tested aminoglycosides (amikacin, 136 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 5 gentamicin, kanamycin, streptomycin, tobramycin), but also to trimethoprim/sulfamethoxazole, 137 aztreonam (monobactam) and ceftazidime (cephalosporin) (Figure 2, Table 2 ) highlighting the 138 crucial role for p1AB5075 towards multi -drug resistance . No difference was observed to 139 ciprofloxacin (fluoroquinolone), cefoperazone, cefepime (cephalosporins), doripenem, imipenem, 140 meropenem (carbapenems), nitrofurantoin (nitrofura n), erythromycin (macrolide), vancomycin 141 (glycopeptide), tetracycline, tigecycline (tetracyclines), cefoxitin (cephamycin), oxacillin, ticarcillin 142 (β-lactams), chloramphenicol and the broad-spectrum β-lactam antibiotic/inhibitor combination 143 piperacillin/tazobactam. We noted a difference in cephalosporin resistance , where wild-type and 144 Δp1AB5075 strains were resistant to cefoperazone and cefepime but not ceftazidime indicating that 145 ceftazidime resistance is mediated by Δp1AB5075 (Figure 2). Resistance to carbapenems did not 146 change which is mediated by a chromosomally encoded oxa-23 gene (Intorcia et al. , 2024) . 147 Resistance to chloramphenicol was not altered either, likely to the presence of additional 148 chloramphenicol resistance genes craA and cpxE (ABUW_0982) (Roca et al., 2009; Karalewitz and 149 Miller, 2018). 150 151 Plasmid-based complementation of resistance genes reveals individual contributions to AMR. 152 As none of the antibiotic resistance genes have been genetically tested for their individual 153 contributions towards antibiotic resistance in A. baumannii AB5075 except for the role of cmlA in 154 resistance to chloramphenicol (de Dios et al. , 2022) , we aimed to characterise seven predicted 155 p1AB5075-encoded antibiotic resistance genes aacA4 (ABUW_RS19275), aadA2 (ABUW_RS19305), 156 aadB (ABUW_RS19315), adh (ABUW_RS19420), strA (ABUW_RS19300), strB (ABUW_RS19295), 157 and blaGES-11 (ABUW_RS19280) by ectopically expressing them from a plasmid in the Δp1AB5075 158 strain. Plasmid-based complementation in A. baumannii routinely utilises the shuttle plasmid (or its 159 origin of replication) pWH1266, which has been assembled from pBR322 and pWH1277 (Hunger et 160 al., 1990). The latter pWH1277 is only partially sequenced, therefore we first sequenced the plasmid 161 pWH1266, which assembled into a plasmid of 8911 bp. To compare resistance levels, all genes were 162 expressed from the same pWH1266-endogenous promoter of the bla gene (Figure 1C) and equipped 163 with the same ribosome binding site (AGGAGG) to ensure equal rate of translation. During cloning, 164 the bla gene of pWH1266 is removed, leaving tetA as the sole (tetracycline) resistance gene. As 165 controls, AB5075 WT and Δp1AB5075 were equipped the pWH1266 “empty” plasmid. Antibiotic 166 disk diffusion assays were performed as before , and the phenotypes in WT and Δp1AB5075 strains 167 remained the same in the presence of pWH1266 compared to the strains without pWH1266 (Figure 168 3, Supplementary Figures 1 & 2). As expected, mild resistance to tetracycline was acquired, which 169 is mediated by tetA located on pWH1266 (Supplementary Figure 2). Typically, 12.5 µg/ml 170 tetracycline is used in liquid broth, therefore, full resistance is not achieved to the 30 µg of the 171 antibiotic-containing disk. Increased aminoglycoside sensitivity was noted upon deletion of 172 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 6 p1AB5075 (Figure 2, Figure 3), and resistance could be restored by expression of either adh (Figure 173 3C), aadB (Figure 3D), aadA2 (Figure 3E), aacC4 (Figure 3F) or strA (Figure 3G). Expression of 174 adh (APH(3’)-VI), which is not part of RI-2 and is flanked by two IS30 family transposases, restored 175 resistance kanamycin and further increased resistance to amikacin in comparison to the wild-type 176 strain (Figure 3A & C). Expression of aadB (ANT(2'')-Ia) restored resistance to kanamycin and 177 increased resistance to gentamicin, and tobramycin (Figure 3 A & D). Expression of aacC4 only 178 partially restored resistance to kanamycin (Figure 3F) and strA provided resistance to streptomycin 179 (Figure 3G). Expression of strB did not restore resistance to any of the tested antibiotics (Figure 180 3H). Therefore, the disk diffusion assays detected genes responsible for p1AB5075-mediated 181 aminoglycoside resistance showing also overlap in providing resistance, where adh, aadB and to a 182 lower extent aacCA4 restored resistance to kanamycin (Figure 3C, D & F), and aadA2 and strA 183 providing resistance to streptomycin (Figure 3E & G). Previously, aacA4 was described as a 184 potential pseudogene (Gallagher et al., 2015), we now show it is functional. The gene outside of RI-185 2, adh, was the only gene providing level resistance to amikacin, while RI-2-encoded aadB was the 186 only gene restoring resistance to gentamicin and tobramycin. Expression of blaGES-11 restored 187 resistance to three antibiotics: ceftazidime, cefepime and aztreonam (Figure 4), which was in line 188 with previous work (Moubareck et al., 2009). 189 To better quantify the contributions of the ARGs to antibiotic resistance and because there 190 was overlap in providing resistance to several antibiotics, minimal inhibitory concentrations were 191 determined by broth microdilution assays for selected antibiotics (Table 3). Expression of ARGs from 192 pWH1266 not only restored resistance, but also routinely increased MICs, likely due to 193 overexpression from the bla promoter of the pWH1266 plasmid (Table 3). Expression of aadB 194 increased the MIC to tobramycin from 32 to 128 μg/ml and to gentamicin from 256 to >4096 μg/ml, 195 adh increased the MIC to amikacin from 512 to 2048 μg/ml, aadA2 and strA increased the MIC to 196 streptomycin from 1024 to >2048 μg/ml (Table 3) matching the data obtained from the disk diffusion 197 assays ( Figure 2 , Table 2). Complementation with pWH1266-aacA4 restored the resistance to 198 tobramycin to wild -type levels (32 μg/ml) but also provided a n increased low-level resistance to 199 multiple aminoglycosides (increases of gentamicin 4-fold, kanamycin 64-fold and amikacin 4-fold) 200 compared to Δp1AB5075 pWH1266 but did not reach wild -type resistance levels. As observed in 201 disk diffusion assays, strB did not recover any of the antibiotic sensitivities. 202 203

204 Multi-drug resistance in A. baumannii is mediated through extensive genetic diversity supported by 205 different resistance islands, plasmids, including p1AB5075, and other mobile genetic elements. Here, 206 we characterised the contribution of p1AB5075 and nine p1AB5075-encoded ARGs towards 207 antibiotic resistance. We observed that loss of p1AB5075 renders A. baumannii AB5075 sensitive to 208 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 7 multiple antibiotics including aminoglycosides, cephalosporins, trimethoprim/sulfamethoxazole and 209 chloramphenicol. We used th is Δp1AB5075 genetic background to genetically dissect the 210 contributions of p1AB5075 -encoded antibiotic resistance genes . As the plasmid p1AB5075 is 211 transferrable from AB5075 to many other A. baumannii and non-baumannii strains, transfer would 212 convert any antibiotic sensitive strain to a MDR strain (Nasser et al., 2024; Martz et al., 2025). We 213 determined that amikacin resistance is predominantly mediated by a gene outside of RI -2 ( adh, 214 (ABUW_RS19420, APH(3')-VI), which is flanked by IS30 family transposases , which have been 215 described to contain ARGs in A. baumannii including the first description of a blaNDM-1 in Poland 216 (Jachowicz-Matczak et al., 2025). However, overexpression of aacA4 increased the MIC to amikacin 217 four-fold, showing that RI -2 amplification through recombination alone can lead to substantial 218 amikacin resistance (Anderson et al., 2020). Indeed, overexpression of aacA4 conferred low level 219 cross resistance to other aminoglycosides (gentamicin, kanamycin and streptomycin) as well , 220 expanding our knowledge of A. baumannii aminoglycoside resistance. Chloramphenicol resistance 221 remained unchanged in the crude disk diffusion assay upon deletion of p1AB5075 , which is in 222 agreement with a previous study (de Dios et al. , 2022) , and is due to the presence of addition 223 chloramphenicol resistance genes craA and cpxE (Karalewitz and Miller, 2018; Foong et al., 2019). 224 To our knowledge, although expected upon loss of blaGES-11 the sensitivity to cephalosporins has not 225 been shown for AB5075, yet. We now confirm experimentally that this is due to the presence of 226 blaGES-11 located in RI -2. AB5075 Δp1AB5075 remains resistant to carbapenems imipenem and 227 meropenem though, due to the presence of an oxa-23 gene on the chromosome. Similar to AB5075, 228 a clinical isolate of A. baumannii isolate from Tunisia with a chromosomally encoded oxa-23 and a 229 plasmid-encoded blaGES-11 copy isolated from a clinical was reported in 2014 (Charfi-Kessis et al., 230 2014). Conjugation of the plasmid carrying blaGES-11 led to increased resistance to ticarcillin, 231 ticarcillin/clavulanic acid, piperacillin, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, 232 aztreonam and even a slight increase to meropenem and imipenem, which might have been masked 233 by the presence of oxa-23 in our strains (Moubareck et al., 2009). A concerning finding was that 234 blaGES-11 may mutate (Gly170Ser) to blaGES-14 resulting in a significant increase in imipenem (from 2 235 to >32 µg/ml) and meropenem (from 4 to 32 µg/ml) resistance as shown in A. baumannii isolated 236 from wound infections of soldiers of Ukraine (Kondratiuk et al., 2025). 237 Apart from the clear role of pAB5075 for AMR in AB5075, future studies may investigate the 238 wider impact on A. baumannii AB5075 biology as the plasmid is transferrable. Other examples have 239 been reported where (loss) of plasmids had profound effects on gene regulation and pathogenesis. 240 The plasmid pAB3 was shown to repress type -6 secretion in A. baumannii ATCC17978 (Weber et 241 al., 2015) and a plasmid-encoded copy of H-NS located on plasmid pAB5 in A. baumannii UPAB1 242 was reported to regulate biofilm formation (Benomar et al., 2021). As p1AB5075 also contains a copy 243 of an H -NS family protein, there is potential that it might mediate similar crosstalk between 244 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 8 p1AB5075- and chromosomally encoded genes. Certainly, a more comprehensive study of plasmids 245 in A. baumannii will be required to better understand their impact on A. baumannii biology beyond 246 AMR. 247 248 FUNDING INFORMATION 249 O. Plunkett received support from Trinity College Dublin (Provost's PhD Project Award 2020/21). 250 251 AUTHOR CONTRIBUTIONS 252 Ideas; formulation or evolution of overarching research goals and aims (OP, KS, CK). Preparation, 253 creation and/or presentation of the published work, specifically visualization/data presentation (OP, 254 CK). Conducting a research and investigation process, specifically performing the experiments, or 255 data/evidence collection (OP, ASE, KS, CK). Oversight and leadership responsibility for the research 256 activity planning and execution, including mentorship external to the core team (KS, CK). Acquisition 257 of the financial support for the project leading to this publication (CK). Preparation, creation and/or 258 presentation of the published work, specifically writing the initial draft (OP, KS, CK). Preparation, 259 creation and/or presentation of the published work by those from the original research group, 260 specifically critical review, commentary or revision – including pre- or post-publication stages (OP, 261 ASE, KS, CK). 262 263

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 11 Valcek, A., Philippe, C., Whiteway, C., Robino, E., Nesporova, K., Bové, M., et al. (2022b) Phenotypic Characterization and Heterogeneity among Modern Clinical Isolates of Acinetobacter baumannii. Microbiology Spectrum 11: e03061-22. Weber, B.S., Ly, P.M., Irwin, J.N., Pukatzki, S., and Feldman, M.F. (2015) A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii. Proceedings of the National Academy of Sciences 112: 9442–9447. Whiteway, C., Breine, A., Philippe, C., and Van der Henst, C. (2022a) Acinetobacter baumannii. Trends in Microbiology 30: 199–200. Whiteway, C., Valcek, A., Philippe, C., Strazisar, M., De Pooter, T., Mateus, I., et al. (2022b) Scarless excision of an insertion sequence restores capsule production and virulence in Acinetobacter baumannii. The ISME Journal 16: 1473–1477. Zhang, Y ., Werling, U., and Edelmann, W. (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Research 40: e55. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 12 Table 1: Annotation of antibiotic resistance genes of p1AB5075. Gene Locus ID Annotation adh ABUW_RS19420 APH(3')-VI family aminoglycoside O-phosphotransferase sul1 ABUW_RS19260 sulphonamide-resistant dihydropteroate synthase qacEΔ1 ABUW_RS19265 quaternary ammonium compound efflux SMR transporter QacE Δ1 dfrA7 ABUW_RS19270 trimethoprim-resistant dihydrofolate reductase DfrA7 aacA4 ABUW_RS19275 aminoglycoside N-acetyltransferase AAC(6')-Ib3 blaGES-11 ABUW_RS19280 Extended-spectrum class A beta-lactamase GES-11 strB ABUW_RS19295 APH(6)-I family aminoglycoside O-phosphotransferase aph(3'')-Ib (strA) ABUW_RS19300 aminoglycoside O-phosphotransferase APH(3'')-Ib aadA2 ABUW_RS19305 ANT(3'')-Ia family aminoglycoside nucleotidyltransferase AadA2 cmlA ABUW_RS19310 CmlA family chloramphenicol efflux MFS transporter aadB ABUW_RS19315 aminoglycoside nucleotidyltransferase ANT(2'')-Ia .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 13 Table 2: Average of inhibition zones of AB5075 mutants measured in mm (n = 3) determined by Kirby -Bauer disk diffusion assays. AK=amikacin, CN=gentamicin, K=kanamycin, S=streptomycin, TOB=tobramycin, TE=tetracycline, TGC=tigecycline, IPM=imipenem, MEM=meropenem, DOR=doripenem, CFP=, CAZ=ceftazidime, FEP=cefepime, ATM=aztreonam, TIC=ticarcillin, OX=oxacillin, FOX=cefoxitin, TZP=piperacillin/tazobactam, CIP=ciprofloxacin, C= chloramphenicol, SXT=trimethoprim/sulfamethoxazole, F=nitrofurantoin, E=erythromycin, V A=vancomycin. Aminoglycosides Tetracyclines β-lactam Others AK 30 CN 30 K 5 S 25 TOB 10 TE 30 TGC 15 IPM 10 MEM 10 DOR 10 CFP 30 CAZ 30 FEP 30 ATM 30 TIC 75 OX 1 FOX 30 TZP 110 CIP 5 C 30 SXT 25 F 100 E 15 VA 30 Wildtype 7 0 0 9 10 24 19 12 9 11 0 0 0 0 0 0 0 0 0 0 0 0 22 0 Δp1AB5075 21 20 17 16 18 22 17 13 10 12 0 19 0 7 0 0 0 0 0 7 25 0 20 0 Wildtype pWH1266 9.5 10 0 12 11 10 18 12 10 12 0 0 0 0 0 0 0 0 0 0 0 0 21 0 Δp1AB5075 pWH1266 24 21 19 15 18 10 19 12 9 12 0 17 18 8 0 0 0 0 0 7 26 0 19 0 Δp1AB5075 pWH1266- aadB 21 0 0 15 0 10 15 14 10 13 0 19 15 10 0 0 0 0 0 10 27 0 20 0 Δp1AB5075 pWH1266- adh 0 23 0 16 19 10 16 12 10 12 0 20 17 9 0 0 0 0 0 9 28 8 20 0 Δp1AB5075 pWH1266- aacA4 21 20 8 16 14 9 18 14 14 14 0 22 19 11 0 0 0 0 0 12 25 7 21 0 Δp1AB5075 pWH1266- aadA2 27 22 19 0 18 9 15 12 10 11 0 18 17 10 0 0 0 0 0 8 29 7 19 0 Δp1AB5075 pWH1266- strA 27 25 18 0 17 11 18 14 10 12 0 18 15 10 0 0 0 0 0 10 30 8 20 0 Δp1AB5075 pWH1266- strB 24 23 17 17 18 10 15 12 10 11 0 20 19 12 0 0 0 0 0 11 28 8 20 0 Δp1AB5075 pWH1266- blaGES-11 23 23 26 12 21 0 17 13 10 12 0 0 0 0 0 0 0 0 0 8 30 8 21 0 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 14 Table 3 : Minimum inhibitory concentration of aminoglycoside antibiotics determined by broth microdilution assay. Values are in μg/ml. CN=gentamicin, K= kanamycin, A K=amikacin, S=streptomycin, TOB= tobramycin, CEF= cefepime, IMP= imipenem, ME M=meropenem. ND=not determined. Strain CN K AK S TOB CEF IMP MEM WT pWH1266 256 > 4096 512 1024 32 256 64 16 Δp1AB5075 pWH1266 8 8 8 256 0.5 32 64 8 Δp1AB5075 pWH1266-aadB > 4096 2048 8 256 128 32 64 8 Δp1AB5075 pWH1266-adh 8 >4096 2048 256 0.5 32 64 8 Δp1AB5075 pWH1266-aadA2 8 16 8 >2048 0.5 32 64 8 Δp1AB5075 pWH1266-aacA4 32 512 32 256 32 32 64 8 Δp1AB5075 pWH1266-strA 8 16 8 >2048 0.5 32 64 8 Δp1AB5075 pWH1266-strB 8 16 8 256 0.5 32 64 8 Δp1AB5075 pWH1266-blaGES-11 ND ND ND ND ND 1024 64 8 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 15 FIGURES Figure 1: Map of A. baumannii AB5075 p1AB5075 (A), Resistance Island -2 (B) and complementation plasmid pWH1266 (C) . (A) Aminoglycoside genes are highlighted in orange, chloramphenicol resistance gene cmlA in pink, sulphonamide resistance gene sul1 in light green, beta- lactamase-encoding blaGES-11 in light blue and trimethoprim resistance gene dfrA7 in purple. The map was created with Proksee (Grant et al., 2023). (B) Linear depiction of p1AB5075-encoded Resistance Island-2. (C) Schematic map of pWH1266 plasmid used for complementation. The bla gene of pWH1266 was replaced by the ARG (blue) resulting of transcription of ARGs from the Pbla promoter (grey, Pbla). All ARGs are translated using the same ribosome binding site (orange, RBSAGGAGG). The tetA gene of pWH1266 confers resistance to tetracycline. The plasmid is not drawn to scale. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 16 Figure 2: Antibiotic disk diffusion assays comparing A. baumannii AB5075 wild-type (WT) and Δp1AB5075 strains . MH2 agar plates were lawned with w ild-type A. baumannii AB5075 and Δp1AB5075 and antibiotic-containing disks were placed on the agar surface . The p lates were incubated for 24 h at 37°C. (A) AK (amikacin 30 µg), SXT (trimethoprim-sulfamethoxazole 25 µg), TOB (tobramycin 10 µg), S (streptomycin 25 µg), CN (gentamicin 30 µg), K (kanamycin 5 µg), (B) nitrofurantoin (F 100 µg), D (doripenem 10 µg), MEM (meropenem 10 µg), V A (vancomycin 20 µg), IPM (imipenem 10 µg), E (erythromycin 15 µg), (C) TGC (tigecycline, 15 µg), TE (tetracycline, 30 µg), OX (oxacillin, 1 µg) , FOX (cefoxitin, 30 µg) , TZP (piperacillin/tazobactam, 110 µg) , TIC (ticarcillin, 75 µg), (D) CAZ (ceftazidime, 30 µg), CFP (cefoperazone, 30 µg) , FEP (cefepime, 30 µg), ATM (aztreonam, 30 µg), C (chloramphenicol, 30 µg), CIP (ciprofloxacin, 5 µg). .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 17 Figure 3: Antibiotic disk diffusion assays comparing A. baumannii AB5075 wild-type (WT, A) and Δp1AB5075 (B) strains carrying pWH1266 or pWH1266 -ARG: pWH1266-adh (C), pWH1266-aadB (D), pWH1266-aadA2 (E), pWH1266-aacA4 (F), pWH1266-strA (G) or pWH1266- strB (H). AK (amikacin 30 µg), SXT (trimethoprim -sulfamethoxazole 25 µg), TOB (tobramycin 10 µg), S (streptomycin 25 µg), CN (gentamicin 30 µg), K (kanamycin 5 µg) . MH2 agar plates containing t etracycline were lawned with w ild-type A. baumannii AB5075 and Δp1AB5075 and antibiotic-containing disks were placed on the agar surface . The plates were incubated for 24 h at 37°C. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint 18 Figure 4: Antibiotic disk diffusion assays comparing A. baumannii AB5075 wild-type (WT, A) and Δp1AB5075 (B) strains carrying pWH1266 or pWH1266-blaGES-11 (C). CAZ (ceftazidime, 30 µg), CFP (cefoperazone, 30 µg) , FEP (cefepime, 30 µg), ATM (aztreonam, 30 µg), C (chloramphenicol, 30 µg), CIP (ciprofloxacin, 5 µg) . MH2 agar plates containing tetracycline were lawned with wild-type A. baumannii AB5075 and Δp1AB5075 and antibiotic-containing disks were placed on the agar surface. The plates were incubated for 24 h at 37°C. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.29.715119doi: bioRxiv preprint