Characterisation of the carbapenem-resistantAcinetobacter baumanniiclinical reference isolate BAL062 (CC2:KL58:OCL1): resistance properties and capsular polysaccharide structure

20 The carbapenem resistant Acinetobacter baumannii isolate BAL062 is a clinical reference 21 isolate used in several recent experimental studies. It is from a ventilator associated 22 pneumonia (VAP) patient in an intensive care unit at the Hospital for Tropical Diseases 23 (HTD), Ho Chi Minh City, Vietnam in 2009. Here, BAL062 was found to belong to the B 24 sub-lineage of global clone 2 (GC2) isolates in the previously reported outbreak (2008 and 25 2012) of carbapenem-resistant VAP A. baumannii at the HTD. While related sub-lineage B 26 outbreak isolates were extensively antibiotic resistant and carry GC2-associated genomic 27 resistance islands, AbGRI1, AbGRI2 and AbGRI3, BAL062 has lost AbGRI3 and three 28 aminoglycoside resistance genes, armA, aacA4 and aphA1, leading to amikacin and 29 kanamycin susceptibility. The location of Tn2008VAR found in the chromosome of this sub-30 lineage was also corrected. Like many of the outbreak isolates, BAL062 carries the KL58 31 gene cluster at the capsular polysaccharide (CPS) synthesis locus and an annotation key is 32 provided. As information about K type is important for development of novel CPS-targeting 33 therapies, the BAL062 K58-type CPS structure was established using NMR spectroscopy. It 34 is most closely related to K2 and K93, sharing similar configurations and linkages between K 35 units and contains the rare higher monosaccharide, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-36 glycero-L-manno-non-2-ulosonic acid (5,7-di-N-acetyl-8-epipseudaminic acid; 37 8ePse5Ac7Ac), the 8-epimer of Pse5Ac7Ac (5,7-di-N-acetylpseudaminic acid). Inspection of 38 publicly available A. baumannii genomes revealed a wide distribution of the KL58 locus in 39 geographically diverse isolates belonging to several sequence types that were recovered over 40 two decades from clinical, animal, and environmental sources. 41 42 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 3 IMPORTANCE 43 Many published experimental studies aimed at developing a clearer understanding of the 44 pathogenicity of carbapenem resistant Acinetobacter baumannii strains currently causing treatment 45 failure due to extensive antibiotic resistance are undertaken using historic, laboratory adapted 46 isolates. However, it is ideal if not imperative that recent clinical isolates are used in such studies. 47 The clinical reference isolate characterized here belongs to the dominant A. baumannii GC2 clone 48 causing extensively resistant infections, and has been used in various recent studies. Correlation of 49 resistance profiles and resistance gene data is key to identifying genes available for gene knockout 50 and complementation analyses, and we have mapped the antibiotic resistance genes to find 51 candidates. Novel therapies, such as bacteriophage or monoclonal antibody therapies, currently 52 under investigation as alternatives or adjuncts to antibiotic treatment to combat difficult-to-treat 53 CRAb infections often exhibit specificity for specific structural epitopes of the capsular 54 polysaccharide (CPS), the outer-most polysaccharide layer. Here, we have solved the structure of 55 the CPS type found in BAL062 and other extensively resistant isolates. As consistent gene naming 56 and annotation are important for locus identification and interpretation of experimental studies, we 57 also have correlated automatic annotations to the standard gene names. 58 59 60 61 62

Acinetobacter baumannii, BAL062, capsular polysaccharide, KL58, 8ePse, 5,7-63 diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid. 64 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 4

65 Carbapenem resistant Acinetobacter baumannii (CRAb) are a leading cause of antibiotic resistant 66 nosocomial infections worldwide (1) and have limited treatment options remaining (2). Hence, 67 alternate therapies are currently being sought. Although other clonal complexes (CC) such as CC1 68 (GC1), CC10, CC25 and CC79 are important, the clonal complex CC2 (also known as Global 69 Clone 2, GC2) that is found on all inhabited continents, accounts for the majority of extensively 70 resistant nosocomial A. baumannii isolates. 71 Owing to concerns about the use of early A. baumannii isolates such as ATCC17978 and 72 ATCC19606 to study the pathogenesis of A. baumannii, particularly that they may be laboratory 73 adapted and hence not strictly representative of current clinical isolates, a number of clinical 74 isolates have begun to be used (3-5). The A. baumannii isolate BAL062 is a clinical carbapenem 75 resistant GC2 isolate that has been utilised for this purpose. BAL062 had been recovered in 2009 76 from a patient with ventilator associated pneumonia (VAP) in an intensive care unit (ICU) at the 77 Hospital for Tropical Diseases (HTD) in Ho Chi Minh City, Vietnam (6). It has since been used to 78 develop a TraDIS library (7) and the complete genome sequence is available (NCBI GenBank 79 accession number LT594095.1; (8)). BAL062 has also been used as a clinical reference isolate in 80 several experimental studies. The BAL062 library has been used to identify genes that contribute to 81 resistance to the last-resort antibiotic colistin (8, 9), and several clinically relevant biocides (7, 10). 82 Additional studies on spermidine/spermine efflux (11), a comparison to other clinical and 83 environmental isolates (12, 13), and demonstration of the utility of novel suicide vectors (14) have 84 also used BAL062. 85 Previously, a series of carbapenem resistant isolates belonging to both GC2 and CC10 were 86 reported to have caused an outbreak between 2008 and 2012 in the same ICU at the HTD in Ho Chi 87 Minh City (15). Most of the carbapenem resistant isolates carried oxa23, the dominant and most 88 widespread gene attributed to the spread of carbapenem resistance (16). However, oxa23 is found in 89 several distinct contexts (16, 17) and, if on the chromosome, their location can be characteristic for 90 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 5 a specific lineage (18). Phylogenetic analysis revealed that the GC2 outbreak isolates could be 91 separated into several distinct sub-lineages, designated A-E, and each sub-lineage had acquired 92 oxa23 independently. Lineage B carried a novel oxa23-containing transposon designated 93 Tn2008VAR (Fig. 1A). Only lineage D carried KL2 at the chromosomal K locus (KL) for 94 biosynthesis of the capsular polysaccharide (CPS) and KL2 was believed to be ancestral. Most 95 lineage E carried KL49, and lineages A-C isolates carried KL58, with a single exception where 96 KL32 had replaced KL58 (15). Recently, four HTD GC2 isolates from this outbreak were compared 97 with ATCC17978 and shown to have increased virulence in mice with systemic dissemination and 98 persistent colonisation of airways (19). This included two isolates with KL58 (BAL084 lineage B; 99 BAL215 lineage C), one with KL2 (BAL276 lineage D) and one with KL49 (BAL191 lineage E), 100 Though the Vietnam GC2 outbreak isolates were resistant to a number of antibiotics, and the 101 oxa23 carbapenem resistance gene was present in different contexts, the remaining antibiotic 102 resistance genes were not examined or reported previously (15). Most carbapenem resistant GC2 103 isolates carry chromosomal islands known as AbGRI1 and AbGRI2 that include genes generally 104 conferring resistance to early antibiotics (20-23). A third, chromosomally-located resistance island, 105 AbGRI3, that includes the armA gene is not found in early GC2 isolates, but is present in many 106 isolates recovered after 2003 (24). The armA gene confers resistance to all clinically relevant 107 aminoglycosides which are used as a last resort to treat carbapenem resistant infections (25). 108 Though not included in the previous study, BAL062 is clearly from the same outbreak 109 granted its place and year of isolation. Here, we have placed BAL062 within one of the specific 110 sub-lineages identified previously, and examined the resistance gene profiles of all the GC2 111 outbreak isolates. BAL062 was found to carry KL58, and we also report the structure of the K58 112 type CPS produced by BAL062. 113 114

115 A. baumannii BAL062 is a multiply antibiotic resistant GC2 isolate 116 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 6 The complete genome sequence of BAL062 (NCBI GenBank accession numbers LT594095.1 117 (chromosome) and LT594096.1 (plasmid)) indicates that it belongs to sequence type (ST) 1550 in 118 the A. baumannii Institut Pasteur (IP) multi-locus sequence typing (MLST) scheme, identifying it as 119 a single locus variant (SLV) of ST2 that represents GC2. This was also noted recently (12). 120 Previously, BAL062 had been recorded as resistant to carbapenems (imipenem), penicillins 121 and β-lactamase inhibitors (piperacillin/tazobactam, ampicillin), fluroquinolones (ofloxacin), third 122 generation cephalosporins (ceftazidime, ceftriaxone, cefepime), aminoglycosides (gentamicin and 123 amikacin), and sulfonamides and trimethoprim (co-trimoxazole) (9). Analysis of antibiotic 124 resistance determinants revealed that resistance to carbapenems was due to the presence of an 125 oxa23 gene (locus tag BAL062_03803) within an unusual Tn2008-like transposon, previously 126 designated Tn2008VAR (15). Tn2008VAR interrupted an acyl-CoA dehydrogenase gene in the 127 chromosome generating a 9 bp target site duplication (Fig. 1A) and this location supersedes the 128 location proposed originally. This transposon was previously found only in the B sub-lineage of the 129 KL58 monophyletic clade (15). An appropriately oriented ISAba1 upstream of the ampC gene 130 (locus tag BAL062_01109) accounts for resistance to third generation cephalosporins. Mutations in 131 the quinolone-determining region of GyrA and ParC explain the fluoroquinolone resistance. 132 However, a determinant for amikacin resistance was not found. 133 The genome also includes strA-strB for spectinomycin resistance and tet(B) for tetracycline 134 resistance, which are both located in an AbGRI1-type island in the comM gene (Fig. 1B). This 135 island is a Tn6022-derived transposon carrying a complete set of transposition genes (tniC-tniA-136 tniB-tniD-tniE). The sul1 (sulfonamide resistance), aadA1 (streptomycin and spectinomycin 137 resistance), and aacC1 (gentamicin resistance) genes are located in an IS26-bounded AbGRI2 type 138 island (Fig. 1B). However, only an IS26 remains of the IS26-bounded AbGRI3 island (Fig. 1B) 139 suggesting that the AbGRI3 resistance genes had been lost during storage of the original isolate. 140 Therefore, the antibiotic susceptibility of BAL062 was re-evaluated using an extended panel 141 of antibiotics. This showed that BAL062 was indeed susceptible to amikacin, as well as to 142 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 7 tobramycin and kanamycin. It was also resistant to tetracycline and further resistant to meropenem 143 and doripenem (carbapenems), ciprofloxacin and nalidixic acid, consistent with the resistance gene 144 profile determined for this isolate. 145 146 A. baumannii BAL062 is a member of GC2:KL58 sub-lineage B 147 The BAL062 genome was found to include the KL58 sequence at the CPS biosynthesis K locus 148 (base positions 3946059 to 3973073) and OCL1 at the OC locus (base positions 587145 to 598627) 149 that determines the outer-core (OC) structure of the lipooligosaccharide. During the HTD outbreak, 150 the KL58 locus had been identified in 29 isolates belonging to either GC2 (n=23) or CC10 (n=6) ( 151 (15); Table S1). To assess the relationship of BAL062 to the GC2:KL58 HTD outbreak isolates, a 152 core-SNP phylogeny was constructed (Fig. 1C). In this phylogeny, BAL062 was positioned within 153 the B sub-lineage, which included four ST2 isolates, BAL056, BAL064, BAL084 and BAL114, 154 that were recovered in the same year (2009) and had been reported to include the Tn2008VAR 155 transposon (15). 156 The additional antibiotic resistance determinants detected were mapped against the tree and, 157 while the other B isolates included many of the resistance genes found in BAL062, consistent with 158 the presence of AbGRI1 and AbGRI2, they also carried armA, aphA1 and aacA4 aminoglycoside 159 resistance genes, as well as blaTEM-1D, mphE-msrE and catB8 genes that were absent from the 160 BAL062 genome (Fig. 1C). This confirmed that the BAL062 isolate currently being used and used 161 to determine the draft (9) and complete (8) genomes had lost the resistance genes expected to be 162 present in AbGRI3 and some of those generally associated with AbGRI2. 163 164 KL58 is related to KL2 and KL93 165 Annotations for the KL58 sequence are available in the BAL114 KL58 sequence record under 166 GenBank accession number KT359617.1, and this sequence is 100% identical (100% coverage) to 167 KL58 in the BAL062 genome (locus tags BAL062_03872-BAL062_03850). As consistent 168 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 8 annotation is key to recognizing the function of genes identified in experimental studies, in Table 1 169 the standard annotations for A. baumannii K loci (26-28) that are used in most publications are 170 compared to those generated using Prokka (29) that appear on the BAL062 genome (LT594095.1) 171 and most automatically annotated genomes. Table 1 also includes the standard and automatic 172 annotations for OCL1 (30, 31). 173 KL58 (Fig. 2) has an arrangement typical of all other sequences found at the K locus in A. 174 baumannii genomes to date (26-28), in that it includes a central region that determines the specific 175 CPS type flanked by a module of wza-wzb-wzc genes for CPS export and galU-pgm genes for 176 synthesis of common sugar precursors. In the previous study, it was reported that the KL58 177 sequence carried by GC2 HTD outbreak isolates in sublineages A-C (Fig. 1C) had likely arisen via 178 a 24 kb sequence replacement involving part of the KL2 locus that was imported from a CC10 179 KL58 isolate (15). The portion shared by KL2 and KL58 (Fig. 2) includes a module of psaABCDEF 180 genes for the synthesis of the monosaccharide 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac), 181 which is a constituent found in the oligosaccharide K-units that make up the K2 CPS (32, 33). 182 The two loci differ in the region that includes predicted glycosyltransferase (gtr) genes and 183 the Wzy polymerase gene for forming glycosidic linkages in the CPS, suggesting that the K2 and 184 K58 structures are composed of similar monosaccharides that are linked together differently. This 185 central portion in KL58 (wzx-gtr9) shares a level of sequence identity (>60% tBLASTx identity) 186 with the A. baumannii KL93 sequence (Fig. 2), and as the K93 structure is related to K2 (34), the 187 K58 structure is likely related to both CPS types. As the structure of the K58 type CPS is unknown, 188 the structure of the CPS produced by BAL062 was determined. 189 190 Monosaccharide composition of CPS recovered from BAL062 191 CPS was isolated from BAL062 cells and purified by Sephadex G-50 Superfine gel 192 chromatography (see methods) for monosaccharide and structural analyses. Sugar analysis of the 193 BAL062 CPS by GLC of the alditol acetates revealed the presence of glucose (Glc), galactose 194 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 9 (Gal), galactosamine (GalN) and a higher order nonulosonate. The presence of signals for N-acetyl 195 groups in the NMR spectra of the CPS (δС 23.0-23.8 (CH3) and 175.5–175.8 (CO), δH 2.00-2.10) 196 indicated that all amino sugars are N-acetylated. Additional chemical analyses on the nonulosonate 197 present revealed the sugar to be the 8-epimer of 5,7-N-acetylpseudaminic acid (Pse5Ac7Ac), known 198 as 8ePse5Ac7Ac or 5,7-N-acetyl-3,5,7,9-tetradeoxynon-2-ulosonic acid. This sugar had only 199 recently been discovered in the CPS of A. baumannii isolate RES-546 that carries the KL135 locus 200 (35) and had not been described for any other isolate to date. 201 202 Structural resolution of the CPS 203 To confirm the order of monosaccharides and overall topology of the BAL062 CPS, the complete 204 structure was established by NMR spectroscopy (Fig. 3) using a set of shift-correlated two-205 dimensional NMR experiments (1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, 206 and1H,13C HMBC). The spin-systems were revealed for the constituent monosaccharides, all being 207 in the pyranose form. The chemical shifts of the monosaccharides are tabulated in Table 2, and the 208 CPS structure is shown in Fig. 4. 209 The chemical shift for C6 of the higher sugar in the CPS ( 73.3 ppm) is similar to the C6 210 chemical shift (73.0 ppm) of -8ePse5Ac7Ac having the axial carboxyl group, but significantly 211 different from that (70.3 ppm) of -8ePse5Ac7Ac with the equatorial carboxyl group (36). 212 Therefore, 8-epipseudaminic acid in the CPS has the axial carboxyl group and is thus -linked. 213 The CPS from BAL062 therefore includes tetrasaccharide K-units with an 8ePse5Ac7Ac-214 (26)-Gal disaccharide branching from a disaccharide main chain composed of D-Glcp and D-215 GalpNAc (Fig. 4A). The attachment of the side chain to position 6 of one of the main-chain 216 components was confirmed by a glycosylation effect, that is a low-field position at 67.8 of the C6 217 signal of the D-Glcp monosaccharide that carries the side chain in the NMR spectra of the CPS, as 218 compared with its position at 62-63 ppm in the spectra of the corresponding non-substituted 219 monosaccharides. 220 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 10 221 Assignment of encoded glycosyltransferases to linkages 222 The composition and topology of the BAL062 CPS is closely related to K2 and K93 types as 223 predicted (Fig. 4A; and see above). As KL58 includes a gene encoding an ItrA2 transferase for 224 initiating CPS synthesis by transferring D-GalpNAc-1P to the lipid carrier (33), and a D-GalpNAc 225 residue is present in the CPS main chain, D-GalpNAc was assigned as the first sugar (Fig. 4A). 226 Hence, the -D-GalpNAc-(1→3)--D-Glcp linkage represents the bond between K-units that is 227 likely formed by the WzyKL58 polymerase (GenPept accession number SBS23904.1) encoded by 228 KL58. Consistent with this conclusion, WzyKL58 shares 84% amino acid (aa) sequence identity with 229 WzyKL93 (34) and 79% aa identity with WzyKL2 (33), both of which form a similar -D-GalpNAc-230 (1→3)--D-Galp linkage in the respective CPS (Fig. 4A). A further search of the BAL062 whole 231 genome sequence did not detect any other Wzy gene candidates, hence WzyKL58 encoded by the K 232 locus was assigned to the -D-GalpNAc-(1→3)--D-Glcp linkage between units in the CPS 233 structure. 234 The three glycosidic linkages in the K-unit are formed by glycosyltransferases encoded by 235 the gtr118, gtr8 and gtr9 genes present in KL58 (Fig. 2). Gtr8 and Gtr9 have previously been found 236 to form the respective linkages in an -D-Galp-(1→6)--D-Glc-(1→3)--D-GalpNAc disaccharide 237 in the K3-type CPS (37, 38). As the same segment is found in the BAL062 structure, Gtr8 and Gtr9 238 were assigned to these linkages (Fig. 4A). Hence, Gtr118 would be responsible for the -239 8ePse5Ac7Ac-(26)-D-Galp linkage in the side chain, and this is supported by Gtr118 sharing 240 82% aa identity with Gtr167 that forms a similar -Pse5Ac7RHb-(26)-D-Galp linkage in the K93 241 CPS (34). 242 243 Distribution of the KL58 locus in A. baumannii genomes 244 In addition to the GC2 (n=23) and CC10 (n=6) KL58 isolates from the HTD outbreak, a search of 245 22,218 A. baumannii genomes available in the NCBI GenBank and non-redundant databases (as of 246 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 11 7th February 2024) identified KL58 in a further 29 isolates (Fig. 5). These included ones from both 247 clinical and environmental sources recovered over a period of two decades (2003 to 2023) from 248 countries including the USA, Canada, China, Singapore, Germany, Poland, and Belgium. Despite 249 the wide distribution, no further isolates from Vietnam or GC2 were detected. However, three 250 isolates from either China (ST10=1; ST574=1) or Belgium (ST574=1) were CC10. The remaining 251 genomes belonged to one of ten other STs or were non-typeable, and included either none or 1-2 252 resistance determinants (Fig. 5). 253 254

255 The use of contemporary nosocomial isolates such as BAL062, over ATCC reference strains 256 isolated >70 years ago, has been recognized by many as key to obtaining data that is relevant to 257 currently circulating clinical isolates (3-5). Although A. baumannii BAL062 is an important clinical 258 GC2 reference isolate as it has been used in multiple experimental studies, some of its basic 259 properties had not been reported. In this study, we report several key properties of the currently 260 available form of this isolate and show that it has lost some of the resistance genes that would 261 account for the phenotype of the original isolate (9). 262 As genetic manipulation relies on techniques that involve resistance markers, suitable strains 263 would ideally be susceptible to one or more appropriate resistance markers. Hence, the previously 264 unnoticed susceptibility to amikacin and kanamycin we have identified will be useful for future 265 studies to replace difficult-to-use resistance markers for selection, including those for tellurite and 266 hygromycin resistance, that are often used. In GC2 isolates, amikacin resistance can be directed by 267 armA or aacA4 located in AbGRI3, and these genes were found in the related lineage B 268 GC2:KL58:OCL1 isolates from the HTD outbreak. However, as only a single IS26 was found in the 269 BAL062 chromosome at this location, it is likely that an IS26-mediated deletion (39) has occurred 270 since BAL062 was first isolated. Likewise, the blaTEM-1D gene and aphA1 kanamycin resistance 271 gene was likely lost via an IS26-mediated deletion internal to AbGRI2 and such events have been 272 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 12 reported previously (20). The loss of resistance markers that were likely present in the original 273 isolate also highlights the importance of continually tracking the properties and potentially the 274 genome sequences of isolates that are being used for experimental studies to ensure that they have 275 not evolved in unexpected ways. 276 We further showed that BAL062 is a member of a discrete lineage of GC2 isolates, referred 277 to as lineage B, from the HTD outbreak (15). A characteristic of this lineage is the presence of a 278 Tn2008VAR transposon carrying oxa23 that interrupts an acyl-CoA dehydrogenase gene in the 279 chromosome. As a complete genome sequence was available for BAL062, we could accurately 280 determine the precise location of the insertion via the identification of a 9 bp target site duplication 281 (TSD) on either side of the transposon (Fig. 1A). This sequence was found to be different to that 282 predicted previously, highlighting the importance of having a complete genome sequence available 283 for clinical reference isolates. 284 Another characteristic of lineage B isolates and BAL062 is the presence of a KL58 sequence 285 at the CPS biosynthesis K locus, which was found to be widely distributed and present in both 286 clinical and environmental isolates. Granted the importance of the CPS and the influence of its 287 specific structure on both virulence (40) and the application of alternate therapies such as 288 monoclonal antibodies (41) and bacteriophage (42, 43), we used BAL062 to determine the K58-289 type structure. The non-2-ulosonic acid component of the K-unit was found to be 8ePse5Ac7Ac, the 290 8-epimer of Pse5Ac7Ac. This sugar was only recently discovered in the A. baumannii K135-type 291 CPS (35), and it was later proposed that the genes responsible for conversion of Pse5Ac7Ac to 292 8ePse5Ac7Ac are located outside the K locus (44). Further work will be needed to identify the 293 genetic determinant(s) for 8ePse5Ac7Ac for the K58 and K135 CPS forms. Nonetheless, the 294 composition and overall topology of the CPS produced by BAL062 was found to be related to the 295 K2 and K93 CPS as expected. 296 The structure correlated with the genetic annotation of KL58 gene cluster using agreed 297 nomenclature is critical to future understanding of its role in or contribution to different phenotypes. 298 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 13 In fact, a previous study that used the BAL062 TraDis library to identify genes involved in 299 susceptibility to or tolerance of colistin (8) showed that that genes at the K locus as well as genes at 300 the OC locus play a role. However, these genes were not identified as being in these locations. 301 Hence, the role of genes involved in synthesis of the outer core of LOS was not noticed. The 302 potential role of CPS in colistin resistance or the involvement of K locus genes in synthesis of the 303 LOS was also neither noticed nor explained, and further work will be needed to explain the role of 304 the genes in colistin resistance. However, the location of genes in the K and OC loci was correctly 305 identified in later studies (10, 11). 306 307

308 Bacterial strain and antibiotic resistance profiling 309 A. baumannii isolate BAL062 was recovered in 2009 from a patient with ventilator associated 310 pneumonia who was admitted to the ICU of the HTD in Ho Chi Minh City, Vietnam (6). The 311 antibiotic resistance profile of BAL062 was determined as described previously (45). 312 313 Bioinformatics analysis 314 The complete genome of BAL062 was downloaded from NCBI assembly accession number 315 GCA_900088705.1 (chromosome: LT594095.1; plasmid: LT594096.1). KL and OCL sequences 316 were identified by command-line Kaptive v 2.0.7 using the current A. baumannii KL (28) and OCL 317 (30) reference sequence databases. BLASTn was used to search 22,218 A. baumannii genomes in 318 the NCBI GenBank and non-redundant databases (available as of 7th February, 2024) for further 319 instances of the KL58 sequence, and the associated metadata (country, collection year and source of 320 isolation) were extracted from corresponding NCBI records and are compiled in Supplementary 321 Table S1. For isolates reported in Schultz et al., draft genome sequences were assembled from short 322 read data (SRA accessions listed in Table S1) using SPAdes (46). 323 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 14 Multilocus sequence typing (MLST) was performed using the A. baumannii Institut Pasteur 324 scheme available at (https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_seqdef). ResFinder v 325 4.4.2 (47) was used to detect antibiotic resistance genes. The core-SNP maximum likelihood 326 phylogeny was constructed using the Bactmap pipeline (https://github.com/nf-core/bactmap) with 327 recombination removed using Gubbins (48), and the tree was visualised using iTOL 328 (https://itol.embl.de/). Figures were created using EasyFig v 2.2.2 (49) and annotated in Adobe 329 Illustrator. 330 331 Isolation of capsular polysaccharide 332 BAL062 was cultivated in 2×TY media overnight. Bacterial cells were harvested by centrifugation 333 (10,000×g, 15 min), washed with and suspended in phosphate buffered saline. The suspension was 334 cooled down to 4 °C, 0.2 volume of CCl3CO2H was added, cells were precipitated by centrifugation 335 (15,000×g, 20 min), and two volumes of acetone were added to the supernatant. After intense 336 shaking, a crude CPS preparation was separated by centrifugation (8,000×g, 20 min), dissolved in 337 water, the pH value was adjusted to pH 8 by adding 1 M NaOH, the CPS was precipitated with 338 acetone and separated by centrifugation as above, dissolved in distilled water and applied to a 339 column (53 × 3.5 cm) of Sephadex G-50 Superfine (Healthcare). Elution was performed with 0.1% 340 HOAc and monitored using a UV-detector (Uvicord, Sweden) at 206 nm to give purified CPS 341 samples. 342 343 Monosaccharide analysis 344 CPS samples (1 mg) were hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were 345 converted conventionally into the alditol acetates analyzed by GLC on a Maestro (Agilent 7820) 346 chromatograph (Interlab, Russia) equipped with an HP-5 column (0.32 mm  30 m) using a 347 temperature program of 160 C (1 min) to 290 C at 7 C min-1. 348 349 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 15 Smith degradation 350 A CPS sample (54 mg) from A. baumannii BAL062 was oxidized with aqueous 0.05 M NaIO4 (1 351 mL) at 20 °C for 48 h in the dark, reduced with an excess of NaBH4 at 20 °C for 16 h. The excess of 352 NaBH4 was destroyed with concentrated AcOH, the solution was evaporated, and the residue was 353 evaporated with methanol (3  1 mL), dissolved in water (in 0.5 mL) and applied to a column (35  354 2 cm) of TSK HW-40. The degraded polysaccharide was eluted with aqueous 0.1% AcOH and 355 hydrolyzed with 2 % HOAc (100 °C, 2 h) to give the -8ePseAc2-(2→1)-Gro glycoside (5.2 mg) 356 and a linear GlcNAc polymer (main-chain polysaccharide, 12 mg), which were isolated by gel-357 permeation chromatography on a column (108  1.2 cm) of TSK HW-40 in 1% HOAc. 358 359 NMR spectroscopy 360 Samples were deuterium-exchanged by freeze-drying from 99.9 % D2O and then examined as 361 solutions in 99.95 % D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz 362 spectrometer (Germany) at 60 °C. Sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (H 0, C 1.6) was 363 used as internal reference for calibration. 2D NMR spectra were obtained using standard Bruker 364 software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. 60-ms 365 MLEV-17 spin-lock time and 150-ms mixing time were used in 1H,1H TOCSY and ROESY 366 experiments, respectively. A 60-ms delay was used for evolution of long-range couplings to 367 optimize 1H,13C HMBC experiments for the coupling constant of JH,C 8 Hz. 1H and 13C chemical 368 shifts were assigned using two-dimensional 1H,1H COSY, 1H,1H TOCSY, and 1H,13C HSQC 369 experiments (Table 2). 370 371

372 We thank A/Prof Amy Cain (Macquarie University, Australia) for providing A. baumannii isolate 373 BAL062, and Dr Stephanie Ambrose (University of Sydney, Australia) for technical assistance. 374 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 16 NMR spectra were recorded in the Department of Structural Studies of N.D. Zelinsky Institute of 375 Organic Chemistry, Moscow. 376 377 Funding 378 This work was supported by the Russian Science Foundation (grant number 19-14-00273), an 379 Australian Research Council (ARC) Future Fellowship (FT230100400) to JJK, and a National 380 Health and Medical Research Council (NHMRC) Investigator grant (GNT1194978) to RMH. 381 382

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Rapid phylogenetic analysis of large samples of recombinant bacterial whole 549 genome sequences using Gubbins. Nucleic Acids Res 43(3):e15. 550 49. Sullivan MJ, Petty NK, Beatson SA. 2011. Easyfig: a genome comparison visualizer. 551 Bioinformatics 27(1):1009-1010. 552 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 23 Figure Legends 553 Figure 1. (A) Tn2008VAR in the BAL062 chromosome (base positions 3893121-3898596). 554 The locus tags indicated on either side are the remnants of the interrupted acyl-CoA 555 dehydrogenase gene in the chromosome. The sequence of the 9 bp target site duplication is 556 shown next to the flags. (B) Genetic arrangement of AbGRIs in BAL062 chromosome: 557 AbGRI1 (base positions 3779179-3801151); AbGRI2 (base positions 2675982-2686299); 558 AbGRI3 (base positions 1400803-1408181). Green boxes indicate insertion sequences, red 559 are resistance genes, orange box is CR2, and flanking chromosomal genes are black. (C) 560 Core-SNP maximum likelihood phylogeny of GC2 genomes carrying KL58 from Vietnam 561 HTB outbreak reported in Schultz et al. (SRA accession numbers listed in Table S1). 562 BAL062 is shown in red. Year of collection, isolation source, STIP and OCL for each isolate 563 are shown next to a presence/absence matrix of antibiotic resistance genes coloured by class. 564 Lineages indicated in Schultz et al. are indicated. 565 566 Figure 2. Comparison of KL58 in the BAL062 chromosome (base positions 3972158-567 3948095) with KL2 from A. baumannii A74 (GenBank accession number KJ459911) and 568 KL93 from A. baumannii B11911 (GenBank accession number CP021345.1; bases 3338181-569 3368604). Genes coloured by function of gene product and grey shading is tBLASTx 570 identity. Colour scheme and scale shown below. 571 572 Figure 3. (A) 13C NMR spectra of the CPS of A. baumannii BAL062. (B) Parts of a two-573 dimensional 1H,13C HSQC spectrum of the CPS of A. baumannii BAL062. The 574 corresponding parts of the one-dimensional 1H and 13C NMR spectra are displayed along the 575 axes. For designations of the monosaccharide residues see Figure 4 and Table 2. 576 577 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 24 578 Figure 4. (A) Structure of the CPS produced by A. baumannii BAL062 compared with K2 579 (32, 33) and K93 (34). (B) Products derived by chemical cleavages of the BAL062 CPS. 580 8ePse5Ac7Ac indicates 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-581 ulosonic acid (di-N-acetyl-8-epipseudaminic acid); Gro indicates glycerol. 582 583 Figure 5. Distribution of KL58 in A. baumannii genome sequences. Colour scheme 584 denoting STs in the Institut Pasteur scheme is shown below. NCBI accession numbers for 585 isolates carrying KL58 are listed in Table S1. 586 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 25 Tables 587 Table 1. Updated gene annotations for the KL58 and OCL1 loci in the BAL062 genome 588 Gene name Locus tag GenPept accession Annotation in LT594095.1 Function/Predicted Function KL58 locus wzc BAL062_03872 SBS23916.1 ptk Protein tyrosine kinase wzb BAL062_03871 SBS23915.1 ptp Low molecular weight protein tyrosine phosphatase wza BAL062_03870 SBS23914.1 Outer membrane protein gna BAL062_03869 SBS23913.1 tuaD_2 UDP-N-acetyl-galactosamine dehydrogenase psaA BAL062_03868 SBS23912.1 capD UDP-N-acetylglucosamine 4,6-dehydratase/5- epimerase psaB BAL062_03867 SBS23911.1 arnB C4-aminotransferase psaC BAL062_03866 SBS23910.1 neuA Cytidylyltransferase psaD BAL062_03865 SBS23909.1 Nucleotidase psaE BAL062_03864 SBS23908.1 N-acetyltransferase psaF BAL062_03863 SBS23907.1 spsE Condensase wzx BAL062_03862 SBS23906.1 Oligosaccharide-unit translocase gtr118 BAL062_03861 SBS23905.1 lst Glycosyltransferase wzy BAL062_03860 SBS23904.1 Oligosaccharide-unit polymerase gtr8 BAL062_03859 SBS23903.1 tagE Glycosyltransferase gtr9 BAL062_03858 SBS23902.1 lsgF Glycosyltransferase itrA2 BAL062_03857 SBS23901.1 wcaJ GalNAc-1P initiating transferase galU BAL062_03856 SBS23900.1 galU UDP-glucose-1-phosphate uridylyltransferase ugd BAL062_03855 SBS23899.1 tuaD_1 UDP-glucose 6-dehydrogenase gpi BAL062_03854 SBS23898.1 pgi glucose-6-phosphate isomerase gne1 BAL062_03853 SBS23897.1 galE_2 UDP-glucose/UDP-N-acetyl-glucosamine 4- epimerase atr42 BAL062_03852 SBS23896.1 Acetyltransferase atr43 BAL062_03851 SBS23895.1 Acetyltransferase pgm BAL062_03850 SBS23894.1 manB Phosphoglucomutase/phosphomannomutase OCL1 locus gtrOC1 BAL062_00583 SBS20708.1 Glycosyltransferase gtrOC2 BAL062_00584 SBS20709.1 Glycosyltransferase pda1 BAL062_00585 SBS20710.1 icaB Polysaccharide deacetylase gtrOC3 BAL062_00586 SBS20711.1 lpsC Glycosyltransferase gtrOC4 BAL062_00587 SBS20712.1 Glycosyltransferase orf1 (ghy) BAL062_00588 SBS20713.1 Unknown gtrOC5 BAL062_00589 SBS20714.1 Glycosyltransferase gtrOC6 BAL062_00590 SBS20715.1 Glycosyltransferase gtrOC7 BAL062_00592 SBS20717.1 sacB Glycosyltransferase .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint 26 Table 2. 1H and 13C NMR chemical shifts (δ, ppm) of the capsular polysaccharide produced 589 by A. baumannii BAL062. 590 Sugar С3 or C1 H3ax,H3eq or H1 С4 or C2 H4 or H2 С5 or C3 H5 or H3 С6 or C4 H6 or H4 С7 or C5 H7 or H5 C8 or C6 H8 or H6 С9 H9 -8ePseAc2 D 37.5 1.62, 2.50 67.8 3.90 49.8 4.18 73.3 3.87 55.0 3.89 67.5 4.37 19.2 1.08 -6)--Gal C 100.0 4.96 70.2 3.83 71.2 3.97 71.3 3.83 71.2 3.99 65.7 3.57, 3.97 -3,6)--Glc B 106.0 4.78 74.7 4.04 85.8 3.91 70.0 4.10 75.6 3.68 67.8 3.74, 3.80 -3)--GalNAc A 103.4 4.51 53.5 3.40 81.5 3.66 70.0 3.59 76.7 3.62 62.9 3.74, 3.95 1H NMR chemical shifts are italicized. 591 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint BAL225 BAL230 BAL315 BAL219 BAL215 BAL295 BAL238 BAL298 BAL128 BAL383 BAL372 BAL369 BAL377 BAL350 BAL341 BAL346 354n BAL339 BAL084 BAL114 BAL056 BAL064 BAL062 UV1897 Tree scale: 0.0001 Lineage B Lineage A Lineage E Lineage C C. 2006 2007 Aminoglycosides 3rd GenerationCephalosporins blaTEM-1D blaPER-1 oxa23 tet(B) tet39 mphE msrE sul2 sul1 catB8 floR armA aphA1 aacC1 aadA1 aadA24 strA/strB Carbapenems Tetracyclines Macrolides Sulfonamides Phenicols Rifamycin Year Source Carriage VAP ST IP aacA4 aadB arr2 aphA6 aac(3)-IId oxa58 2009 VAP 2009 VAP 2009 VAP 2009 VAP 2009 VAP 2011 VAP 2011 VAP 2011 VAP 2011 VAP 2012 VAP 2012 VAP 2012 VAP 2010 VAP 2011 VAP 2010 VAP 2011 VAP 2010 2010 VAP VAP 2011 VAP 2010 VAP 2010 VAP Isolate 2 1550 2 2 2 2 571 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2012 VAP OCL 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 Tn2008VAR A. AbGRI2 AbGRI3 ΔcomMtniC tniA tniB tniD tniE orf uspA sup orf4 tetR(B)tetA(B) rcr2 strB strA orf4b AbGRI1 orf IS26 orfΔ orf orf B. ΔcomM (CACCGATCC) BAL062_03807 BAL062_03800 ISAba33 ISAba1oxa23 (CACCGATCC) (CTCATCCT) orf asr IS26 IS26tnpAΔtnpRtnpMintI1aacC1PaadA1sul1orf5 PQIS6100Δ qacEdelta1 (CGCCAACT) Δ10,253 bp Δ1,156 bp .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint The K locus (KL) CMP-Pse5Ac7Ac synthesisCPS export Simple sugar synthesis KL58 gtr9gtr8 atr42wzc wzb wza gna psaDpsaEpsaA psaB psaC psaF wzx wzyKL58gtr118 itrA2 galU gpi gne1ugd pgmatr43 REGION 1 REGION 2 REGION 3 gtr5gtr4wzc wzb wza gna psaDpsaEpsaA psaB psaC psaF wzx wzyKL2kpsS1 itrA2 galU gpi gne1ugd pgmgtr3 KL2 1 kb Capsule export machinery Nucleotide-sugar biosynthesis Acetyl or acyl transferase (atr) Repeat unit processing Glycosyltransferase (gtr) Initiating transferase (itr) Simple sugar synthesis Other 60% 100% tBLASTx % identity gtr5gtr25wzc wzb wza gna psaG psaHpsaA psaB psaC psaF wzx wzyKL93gtr167 itrA2 galU gpi gne1ugd pgmpgt KL93 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint A. B. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint →3)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→ WzyKL58 [ItrA2] Gtr9 6) Gtr118 (1 Gtr8 → CPS BAL062 WzyKL58 α-8ePse5Ac7Ac-(2→6)-α-D-Galp →3)-β-D-Galp-(1→3)-β-D-GalpNAc-(1→ [ItrA2] Gtr5 Gtr4 6) α-Pse5Ac7Ac-(2→6)-β-D-Glcp (1 →KpsS1 K2 →3)-β-D-Galp-(1→3)-β-D-GalpNAc-(1→ [ItrA2] 6) Gtr5 β-Pse5Ac7R-(2→6)-α-D-Galp (1 Gtr25 →Gtr167 K93 WzyKL2WzyKL2 WzyKL93WzyKL93 AB CD →3)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→ Mainchain polysaccharide AB 8ePse5Ac7Ac-(2→1)-Gro D Glycoside 1 α-8ePse5Ac7Ac-(2→6)-α-D-Galp-(1→6)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→ Tetrasaccharide 2 ABCD A. B. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint IHIT35900 PUMA0184 PUMA0214 98_E23.3 16-Klo_64-1 PUMA0123 LWSM-0248 WU_MDCI_Ab184 17-Lo_4-1 29D2 2023EL-00143 2023EL-00144 2022JQ-00544 2021CK-01311 MRSN1339 MRSN31468 MRSN7782 Ab-69 AB179_VUB SH37 350_n 344_an 341_c 316_an 277_ax UV_1268 BAL383 BAL372 BAL369 BAL339 BAL350 BAL346 BAL341 BAL315 BAL298 BAL295 BAL238 BAL230 BAL225 BAL219 BAL215 BAL128 BAL114 BAL084 BAL064 BAL062 BAL056 UV_1897 354_n Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam China Belgium China USA Germany USA USA USA USA USA Poland Germany USA Germany Singapore Poland Germany Singapore Singapore USA 2010 2006 2007 2009 2009 2009 2009 2009 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2005 2005 2006 2006 2006 2006 2010 2017 2020 2006 2003 2010 2021 2022 2023 2023 2014 2017 2018 2019 2023 2016 2019 2023 2023 2014 blaTEM-1D blaPER-1 blaCARB-1 oxa23 tet(B) tet39 mphE msrE sul2 sul1 catB8 floR armA aphA1 aacC1 aadA1 aadA24 strA/strB Strain Country Year Ab11 Ab9 Ab14 PUMA0140 WU_MDCI_Ab140 MST-SNC-9 PUMA0145 Ab182 PUMA0099 Singapore USA Singapore Canada USA Singapore NA NA NA 2023 2018 2023 NA 2018 2023 NA NA NA Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Source Carriage Connective tissue infection Agricultural surface water VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP VAP Carriage Carriage Carriage Carriage Carriage NA Sputum Urine Wound Groin Surveillance Rectal swab Blood Blood Catheter tip White stork Nestling Earthworm Respiratory tract Pig production setting NA White stork Boot swab NA NA Lizard faeces NA Urine NA NA NA NA NA ST IP ST2 ST1550 ST571 ST574 ST10 ST150 ST154 ST309 ST858 ST342 ST1220 ST1301 ST2373 ST2561 ST2643 Non-typeable aacA4 aadB arr2 aphA6 aac(3)-IId oxa58 tet(X6) aph(4)-Ia aac(3)-IV OCL 1 1 1 1 6 6 6 6 2 1 1 1 2 1 1 2 2 2 2 2 2 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 1 1 1 1 1 1 1 2 3 1 GC2/CC2 CC10 Other BAL377 2012Vietnam VAP 1 Aminoglycosides 3rd GenerationCephalosporins Carbapenems Tetracyclines Macrolides Sulfonamides Phenicols Rifamycin .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint