{"paper_id":"a820b4e9-ae35-47e3-ab1b-65aa29896127","body_text":"1 \nCharacterisation of the carbapenem-resistant Acinetobacter baumannii 1 \nclinical reference isolate BAL062 (CC2:KL58:OCL1): resistance properties 2 \nand capsular polysaccharide structure  3 \n 4 \nAlexander S. Shashkov,1 Nikolay P. Arbatsky,1 Sof’ya N. Senchenkova,1 Andrei S. 5 \nDmitrenok,1 Mikhail M. Shneider,2 Yuriy A. Knirel,1,† Ruth M. Hall,3,† Johanna J. Kenyon4,5,†,* 6 \n 7 \n1N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia 8 \n2 M. M. Shemyakin & Y. A Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of 9 \nSciences, Moscow, Russia 10 \n3 School of Life and Environmental Science, The University of Sydney, Sydney, Australia 11 \n4 Centre for Immunology and Infection Control, School of Biomedical Sciences, Faculty of 12 \nHealth, Queensland University of Technology, Brisbane, Australia 13 \n5 School of Pharmacy and Medical Sciences, Health Group, Griffith University, Gold Coast, 14 \nAustralia 15 \n 16 \n† Authors contributed equally 17 \n* Corresponding author. Tel.: +61 7 5552 9273; E-mail:  j.kenyon@griffith.edu.au 18 \n  19 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 2 \nABSTRACT 20 \nThe carbapenem resistant Acinetobacter baumannii isolate BAL062 is a clinical reference 21 \nisolate used in several recent experimental studies. It is from a ventilator associated 22 \npneumonia (VAP) patient in an intensive care unit at the Hospital for Tropical Diseases 23 \n(HTD), Ho Chi Minh City, Vietnam in 2009. Here, BAL062 was found to belong to the B 24 \nsub-lineage of global clone 2 (GC2) isolates in the previously reported outbreak (2008 and 25 \n2012) of carbapenem-resistant VAP A. baumannii at the HTD. While related sub-lineage B 26 \noutbreak isolates were extensively antibiotic resistant and carry GC2-associated genomic 27 \nresistance islands, AbGRI1, AbGRI2 and AbGRI3, BAL062 has lost AbGRI3 and three 28 \naminoglycoside resistance genes, armA, aacA4 and aphA1, leading to amikacin and 29 \nkanamycin susceptibility. The location of Tn2008VAR found in the chromosome of this sub-30 \nlineage was also corrected.  Like many of the outbreak isolates, BAL062 carries the KL58 31 \ngene cluster at the capsular polysaccharide (CPS) synthesis locus and an annotation key is 32 \nprovided. As information about K type is important for development of novel CPS-targeting 33 \ntherapies, the BAL062 K58-type CPS structure was established using NMR spectroscopy. It 34 \nis most closely related to K2 and K93, sharing similar configurations and linkages between K 35 \nunits and contains the rare higher monosaccharide, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-36 \nglycero-L-manno-non-2-ulosonic acid (5,7-di-N-acetyl-8-epipseudaminic acid; 37 \n8ePse5Ac7Ac), the 8-epimer of Pse5Ac7Ac (5,7-di-N-acetylpseudaminic acid). Inspection of 38 \npublicly available A. baumannii genomes revealed a wide distribution of the KL58 locus in 39 \ngeographically diverse isolates belonging to several sequence types that were recovered over 40 \ntwo decades from clinical, animal, and environmental sources.   41 \n 42 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 3 \nIMPORTANCE 43 \nMany published experimental studies aimed at developing a clearer understanding of the 44 \npathogenicity of carbapenem resistant Acinetobacter baumannii strains currently causing treatment 45 \nfailure due to extensive antibiotic resistance are undertaken using historic, laboratory adapted 46 \nisolates. However, it is ideal if not imperative that recent clinical isolates are used in such studies. 47 \nThe clinical reference isolate characterized here belongs to the dominant A. baumannii GC2 clone 48 \ncausing extensively resistant infections, and has been used in various recent studies. Correlation of 49 \nresistance profiles and resistance gene data is key to identifying genes available for gene knockout 50 \nand complementation analyses, and we have mapped the antibiotic resistance genes to find 51 \ncandidates. Novel therapies, such as bacteriophage or monoclonal antibody therapies, currently 52 \nunder investigation as alternatives or adjuncts to antibiotic treatment to combat difficult-to-treat 53 \nCRAb infections often exhibit specificity for specific structural epitopes of the capsular 54 \npolysaccharide (CPS), the outer-most polysaccharide layer. Here, we have solved the structure of 55 \nthe CPS type found in BAL062 and other extensively resistant isolates. As consistent gene naming 56 \nand annotation are important for locus identification and interpretation of experimental studies, we 57 \nalso have correlated automatic annotations to the standard gene names. 58 \n 59 \n 60 \n 61 \n 62 \nKeywords: Acinetobacter baumannii, BAL062, capsular polysaccharide, KL58, 8ePse, 5,7-63 \ndiacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid.  64 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 4 \nINTRODUCTION 65 \nCarbapenem resistant Acinetobacter baumannii (CRAb) are a leading cause of antibiotic resistant 66 \nnosocomial infections worldwide (1) and have limited treatment options remaining (2). Hence, 67 \nalternate therapies are currently being sought. Although other clonal complexes (CC) such as CC1 68 \n(GC1), CC10, CC25 and CC79 are important, the clonal complex CC2 (also known as Global 69 \nClone 2, GC2) that is found on all inhabited continents, accounts for the majority of extensively 70 \nresistant nosocomial A. baumannii isolates.  71 \nOwing to concerns about the use of early A. baumannii isolates such as ATCC17978 and 72 \nATCC19606 to study the pathogenesis of A. baumannii, particularly that they may be laboratory 73 \nadapted and hence not strictly representative of current clinical isolates, a number of clinical 74 \nisolates have begun to be used (3-5). The A. baumannii isolate BAL062 is a clinical carbapenem 75 \nresistant GC2 isolate that has been utilised for this purpose. BAL062 had been recovered in 2009 76 \nfrom a patient with ventilator associated pneumonia (VAP) in an intensive care unit (ICU) at the 77 \nHospital for Tropical Diseases (HTD) in Ho Chi Minh City, Vietnam (6). It has since been used to 78 \ndevelop a TraDIS library (7) and the complete genome sequence is available (NCBI GenBank 79 \naccession number LT594095.1; (8)). BAL062 has also been used as a clinical reference isolate in 80 \nseveral experimental studies. The BAL062 library has been used to identify genes that contribute to 81 \nresistance to the last-resort antibiotic colistin (8, 9), and several clinically relevant biocides (7, 10). 82 \nAdditional studies on spermidine/spermine efflux (11), a comparison to other clinical and 83 \nenvironmental isolates (12, 13), and demonstration of the utility of novel suicide vectors (14) have 84 \nalso used BAL062. 85 \nPreviously, a series of carbapenem resistant isolates belonging to both GC2 and CC10 were 86 \nreported to have caused an outbreak between 2008 and 2012 in the same ICU at the HTD in Ho Chi 87 \nMinh City (15). Most of the carbapenem resistant isolates carried oxa23, the dominant and most 88 \nwidespread gene attributed to the spread of carbapenem resistance (16). However, oxa23 is found in 89 \nseveral distinct contexts (16, 17) and, if on the chromosome, their location can be characteristic for 90 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 5 \na specific lineage (18). Phylogenetic analysis revealed that the GC2 outbreak isolates could be 91 \nseparated into several distinct sub-lineages, designated A-E, and each sub-lineage had acquired 92 \noxa23 independently. Lineage B carried a novel oxa23-containing transposon designated 93 \nTn2008VAR (Fig. 1A). Only lineage D carried KL2 at the chromosomal K locus (KL) for 94 \nbiosynthesis of the capsular polysaccharide (CPS) and KL2 was believed to be ancestral. Most 95 \nlineage E carried KL49, and lineages A-C isolates carried KL58, with a single exception where 96 \nKL32 had replaced KL58 (15). Recently, four HTD GC2 isolates from this outbreak were compared 97 \nwith ATCC17978 and shown to have increased virulence in mice with systemic dissemination and 98 \npersistent colonisation of airways (19). This included two isolates with KL58 (BAL084 lineage B; 99 \nBAL215 lineage C), one with KL2 (BAL276 lineage D) and one with KL49 (BAL191 lineage E),  100 \nThough the Vietnam GC2 outbreak isolates were resistant to a number of antibiotics, and the 101 \noxa23 carbapenem resistance gene was present in different contexts, the remaining antibiotic 102 \nresistance genes were not examined or reported previously (15). Most carbapenem resistant GC2 103 \nisolates carry chromosomal islands known as AbGRI1 and AbGRI2 that include genes generally 104 \nconferring resistance to early antibiotics (20-23). A third, chromosomally-located resistance island, 105 \nAbGRI3, that includes the armA gene is not found in early GC2 isolates, but is present in many 106 \nisolates recovered after 2003 (24). The armA gene confers resistance to all clinically relevant 107 \naminoglycosides which are used as a last resort to treat carbapenem resistant infections (25). 108 \nThough not included in the previous study, BAL062 is clearly from the same outbreak 109 \ngranted its place and year of isolation. Here, we have placed BAL062 within one of the specific 110 \nsub-lineages identified previously, and examined the resistance gene profiles of all the GC2 111 \noutbreak isolates. BAL062 was found to carry KL58, and we also report the structure of the K58 112 \ntype CPS produced by BAL062.  113 \n 114 \nResults 115 \nA. baumannii BAL062 is a multiply antibiotic resistant GC2 isolate  116 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 6 \nThe complete genome sequence of BAL062 (NCBI GenBank accession numbers LT594095.1 117 \n(chromosome) and LT594096.1 (plasmid)) indicates that it belongs to sequence type (ST) 1550 in 118 \nthe A. baumannii Institut Pasteur (IP) multi-locus sequence typing (MLST) scheme, identifying it as 119 \na single locus variant (SLV) of ST2 that represents GC2. This was also noted recently (12).  120 \nPreviously, BAL062 had been recorded as resistant to carbapenems (imipenem), penicillins 121 \nand β-lactamase inhibitors (piperacillin/tazobactam, ampicillin), fluroquinolones (ofloxacin), third 122 \ngeneration cephalosporins (ceftazidime, ceftriaxone, cefepime), aminoglycosides (gentamicin and 123 \namikacin), and sulfonamides and trimethoprim (co-trimoxazole) (9). Analysis of antibiotic 124 \nresistance determinants revealed that resistance to carbapenems was due to the presence of an 125 \noxa23 gene (locus tag BAL062_03803) within an unusual Tn2008-like transposon, previously 126 \ndesignated Tn2008VAR (15). Tn2008VAR interrupted an acyl-CoA dehydrogenase gene in the 127 \nchromosome generating a 9 bp target site duplication (Fig. 1A) and this location supersedes the 128 \nlocation proposed originally. This transposon was previously found only in the B sub-lineage of the 129 \nKL58 monophyletic clade (15). An appropriately oriented ISAba1 upstream of the ampC gene 130 \n(locus tag BAL062_01109) accounts for resistance to third generation cephalosporins. Mutations in 131 \nthe quinolone-determining region of GyrA and ParC explain the fluoroquinolone resistance. 132 \nHowever, a determinant for amikacin resistance was not found. 133 \nThe genome also includes strA-strB for spectinomycin resistance and tet(B) for tetracycline 134 \nresistance, which are both located in an AbGRI1-type island in the comM gene (Fig. 1B). This 135 \nisland is a Tn6022-derived transposon carrying a complete set of transposition genes (tniC-tniA-136 \ntniB-tniD-tniE). The sul1 (sulfonamide resistance), aadA1 (streptomycin and spectinomycin 137 \nresistance), and aacC1 (gentamicin resistance) genes are located in an IS26-bounded AbGRI2 type 138 \nisland (Fig. 1B). However, only an IS26 remains of the IS26-bounded AbGRI3 island (Fig. 1B) 139 \nsuggesting that the AbGRI3 resistance genes had been lost during storage of the original isolate.  140 \nTherefore, the antibiotic susceptibility of BAL062 was re-evaluated using an extended panel 141 \nof antibiotics. This showed that BAL062 was indeed susceptible to amikacin, as well as to 142 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 7 \ntobramycin and kanamycin. It was also resistant to tetracycline and further resistant to meropenem 143 \nand doripenem (carbapenems), ciprofloxacin and nalidixic acid, consistent with the resistance gene 144 \nprofile determined for this isolate.   145 \n 146 \nA. baumannii BAL062 is a member of GC2:KL58 sub-lineage B  147 \nThe BAL062 genome was found to include the KL58 sequence at the CPS biosynthesis K locus 148 \n(base positions 3946059 to 3973073) and OCL1 at the OC locus (base positions 587145 to 598627) 149 \nthat determines the outer-core (OC) structure of the lipooligosaccharide. During the HTD outbreak, 150 \nthe KL58 locus had been identified in 29 isolates belonging to either GC2 (n=23) or CC10 (n=6) ( 151 \n(15); Table S1). To assess the relationship of BAL062 to the GC2:KL58 HTD outbreak isolates, a 152 \ncore-SNP phylogeny was constructed (Fig. 1C). In this phylogeny, BAL062 was positioned within 153 \nthe B sub-lineage, which included four ST2 isolates, BAL056, BAL064, BAL084 and BAL114, 154 \nthat were recovered in the same year (2009) and had been reported to include the Tn2008VAR 155 \ntransposon (15).  156 \nThe additional antibiotic resistance determinants detected were mapped against the tree and, 157 \nwhile the other B isolates included many of the resistance genes found in BAL062, consistent with 158 \nthe presence of AbGRI1 and AbGRI2, they also carried armA, aphA1 and aacA4 aminoglycoside 159 \nresistance genes, as well as blaTEM-1D, mphE-msrE and catB8 genes that were absent from the 160 \nBAL062 genome (Fig. 1C). This confirmed that the BAL062 isolate currently being used and used 161 \nto determine the draft (9) and complete (8) genomes had lost the resistance genes expected to be 162 \npresent in AbGRI3 and some of those generally associated with AbGRI2. 163 \n 164 \nKL58 is related to KL2 and KL93 165 \nAnnotations for the KL58 sequence are available in the BAL114 KL58 sequence record under 166 \nGenBank accession number KT359617.1, and this sequence is 100% identical (100% coverage) to 167 \nKL58 in the BAL062 genome (locus tags BAL062_03872-BAL062_03850). As consistent 168 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 8 \nannotation is key to recognizing the function of genes identified in experimental studies, in Table 1 169 \nthe standard annotations for A. baumannii K loci (26-28) that are used in most publications are 170 \ncompared to those generated using Prokka (29) that appear on the BAL062 genome (LT594095.1) 171 \nand most automatically annotated genomes. Table 1 also includes the standard and automatic 172 \nannotations for OCL1 (30, 31).  173 \n KL58 (Fig. 2) has an arrangement typical of all other sequences found at the K locus in A. 174 \nbaumannii genomes to date (26-28), in that it includes a central region that determines the specific 175 \nCPS type flanked by a module of wza-wzb-wzc genes for CPS export and galU-pgm genes for 176 \nsynthesis of common sugar precursors. In the previous study, it was reported that the KL58 177 \nsequence carried by GC2 HTD outbreak isolates in sublineages A-C (Fig. 1C) had likely arisen via 178 \na 24 kb sequence replacement involving part of the KL2 locus that was imported from a CC10 179 \nKL58 isolate (15). The portion shared by KL2 and KL58 (Fig. 2) includes a module of psaABCDEF 180 \ngenes for the synthesis of the monosaccharide 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac), 181 \nwhich is a constituent found in the oligosaccharide K-units that make up the K2 CPS (32, 33).  182 \nThe two loci differ in the region that includes predicted glycosyltransferase (gtr) genes and 183 \nthe Wzy polymerase gene for forming glycosidic linkages in the CPS, suggesting that the K2 and 184 \nK58 structures are composed of similar monosaccharides that are linked together differently. This 185 \ncentral portion in KL58 (wzx-gtr9) shares a level of sequence identity (>60% tBLASTx identity) 186 \nwith the A. baumannii KL93 sequence (Fig. 2), and as the K93 structure is related to K2 (34), the 187 \nK58 structure is likely related to both CPS types. As the structure of the K58 type CPS is unknown, 188 \nthe structure of the CPS produced by BAL062 was determined.  189 \n 190 \nMonosaccharide composition of CPS recovered from BAL062 191 \nCPS was isolated from BAL062 cells and purified by Sephadex G-50 Superfine gel 192 \nchromatography (see methods) for monosaccharide and structural analyses. Sugar analysis of the 193 \nBAL062 CPS by GLC of the alditol acetates revealed the presence of glucose (Glc), galactose 194 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 9 \n(Gal), galactosamine (GalN) and a higher order nonulosonate. The presence of signals for N-acetyl 195 \ngroups 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 \nindicated that all amino sugars are N-acetylated. Additional chemical analyses on the nonulosonate 197 \npresent revealed the sugar to be the 8-epimer of 5,7-N-acetylpseudaminic acid (Pse5Ac7Ac), known 198 \nas 8ePse5Ac7Ac or 5,7-N-acetyl-3,5,7,9-tetradeoxynon-2-ulosonic acid. This sugar had only 199 \nrecently been discovered in the CPS of A. baumannii isolate RES-546 that carries the KL135 locus 200 \n(35) and had not been described for any other isolate to date.  201 \n 202 \nStructural resolution of the CPS  203 \nTo confirm the order of monosaccharides and overall topology of the BAL062 CPS, the complete 204 \nstructure was established by NMR spectroscopy (Fig. 3) using a set of shift-correlated two-205 \ndimensional NMR experiments (1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, 206 \nand1H,13C HMBC). The spin-systems were revealed for the constituent monosaccharides, all being 207 \nin the pyranose form. The chemical shifts of the monosaccharides are tabulated in Table 2, and the 208 \nCPS structure is shown in Fig. 4. 209 \nThe chemical shift for C6 of the higher sugar in the CPS ( 73.3 ppm) is similar to the C6 210 \nchemical shift (73.0 ppm) of -8ePse5Ac7Ac having the axial carboxyl group, but significantly 211 \ndifferent from that (70.3 ppm) of -8ePse5Ac7Ac with the equatorial carboxyl group (36). 212 \nTherefore, 8-epipseudaminic acid in the CPS has the axial carboxyl group and is thus -linked. 213 \nThe CPS from BAL062 therefore includes tetrasaccharide K-units with an 8ePse5Ac7Ac-214 \n(26)-Gal disaccharide branching from a disaccharide main chain composed of D-Glcp and D-215 \nGalpNAc (Fig. 4A). The attachment of the side chain to position 6 of one of the main-chain 216 \ncomponents was confirmed by a glycosylation effect, that is a low-field position at 67.8 of the C6 217 \nsignal of the D-Glcp monosaccharide that carries the side chain in the NMR spectra of the CPS, as 218 \ncompared with its position at 62-63 ppm in the spectra of the corresponding non-substituted 219 \nmonosaccharides.  220 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 10 \n 221 \nAssignment of encoded glycosyltransferases to linkages  222 \nThe composition and topology of the BAL062 CPS is closely related to K2 and K93 types as 223 \npredicted (Fig. 4A; and see above). As KL58 includes a gene encoding an ItrA2 transferase for 224 \ninitiating CPS synthesis by transferring D-GalpNAc-1P to the lipid carrier (33), and a D-GalpNAc 225 \nresidue is present in the CPS main chain, D-GalpNAc was assigned as the first sugar (Fig. 4A). 226 \nHence, the -D-GalpNAc-(1→3)--D-Glcp linkage represents the bond between K-units that is 227 \nlikely formed by the WzyKL58 polymerase (GenPept accession number SBS23904.1) encoded by 228 \nKL58. Consistent with this conclusion, WzyKL58 shares 84% amino acid (aa) sequence identity with 229 \nWzyKL93 (34) and 79% aa identity with WzyKL2 (33), both of which form a similar -D-GalpNAc-230 \n(1→3)--D-Galp linkage in the respective CPS (Fig. 4A). A further search of the BAL062 whole 231 \ngenome sequence did not detect any other Wzy gene candidates, hence WzyKL58 encoded by the K 232 \nlocus was assigned to the -D-GalpNAc-(1→3)--D-Glcp linkage between units in the CPS 233 \nstructure. 234 \nThe three glycosidic linkages in the K-unit are formed by glycosyltransferases encoded by 235 \nthe gtr118, gtr8 and gtr9 genes present in KL58 (Fig. 2). Gtr8 and Gtr9 have previously been found 236 \nto form the respective linkages in an -D-Galp-(1→6)--D-Glc-(1→3)--D-GalpNAc disaccharide 237 \nin the K3-type CPS (37, 38). As the same segment is found in the BAL062 structure, Gtr8 and Gtr9 238 \nwere assigned to these linkages (Fig. 4A). Hence, Gtr118 would be responsible for the -239 \n8ePse5Ac7Ac-(26)-D-Galp linkage in the side chain, and this is supported by Gtr118 sharing 240 \n82% aa identity with Gtr167 that forms a similar -Pse5Ac7RHb-(26)-D-Galp linkage in the K93 241 \nCPS (34). 242 \n  243 \nDistribution of the KL58 locus in A. baumannii genomes 244 \nIn addition to the GC2 (n=23) and CC10 (n=6) KL58 isolates from the HTD outbreak, a search of 245 \n22,218 A. baumannii genomes available in the NCBI GenBank and non-redundant databases (as of 246 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 11 \n7th February 2024) identified KL58 in a further 29 isolates (Fig. 5). These included ones from both 247 \nclinical and environmental sources recovered over a period of two decades (2003 to 2023) from 248 \ncountries including the USA, Canada, China, Singapore, Germany, Poland, and Belgium. Despite 249 \nthe wide distribution, no further isolates from Vietnam or GC2 were detected. However, three 250 \nisolates from either China (ST10=1; ST574=1) or Belgium (ST574=1) were CC10. The remaining 251 \ngenomes belonged to one of ten other STs or were non-typeable, and included either none or 1-2 252 \nresistance determinants (Fig. 5).  253 \n 254 \nDISCUSSION 255 \nThe use of contemporary nosocomial isolates such as BAL062, over ATCC reference strains 256 \nisolated >70 years ago, has been recognized by many as key to obtaining data that is relevant to 257 \ncurrently circulating clinical isolates (3-5). Although A. baumannii BAL062 is an important clinical 258 \nGC2 reference isolate as it has been used in multiple experimental studies, some of its basic 259 \nproperties had not been reported. In this study, we report several key properties of the currently 260 \navailable form of this isolate and show that it has lost some of the resistance genes that would 261 \naccount for the phenotype of the original isolate (9).  262 \nAs genetic manipulation relies on techniques that involve resistance markers, suitable strains 263 \nwould ideally be susceptible to one or more appropriate resistance markers. Hence, the previously 264 \nunnoticed susceptibility to amikacin and kanamycin we have identified will be useful for future 265 \nstudies to replace difficult-to-use resistance markers for selection, including those for tellurite and 266 \nhygromycin resistance, that are often used. In GC2 isolates, amikacin resistance can be directed by 267 \narmA or aacA4 located in AbGRI3, and these genes were found in the related lineage B 268 \nGC2:KL58:OCL1 isolates from the HTD outbreak. However, as only a single IS26 was found in the 269 \nBAL062 chromosome at this location, it is likely that an IS26-mediated deletion (39) has occurred 270 \nsince BAL062 was first isolated. Likewise, the blaTEM-1D gene and aphA1 kanamycin resistance 271 \ngene was likely lost via an IS26-mediated deletion internal to AbGRI2 and such events have been 272 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 12 \nreported previously (20). The loss of resistance markers that were likely present in the original 273 \nisolate also highlights the importance of continually tracking the properties and potentially the 274 \ngenome sequences of isolates that are being used for experimental studies to ensure that they have 275 \nnot evolved in unexpected ways.  276 \nWe further showed that BAL062 is a member of a discrete lineage of GC2 isolates, referred 277 \nto as lineage B, from the HTD outbreak (15). A characteristic of this lineage is the presence of a 278 \nTn2008VAR transposon carrying oxa23 that interrupts an acyl-CoA dehydrogenase gene in the 279 \nchromosome. As a complete genome sequence was available for BAL062, we could accurately 280 \ndetermine the precise location of the insertion via the identification of a 9 bp target site duplication 281 \n(TSD) on either side of the transposon (Fig. 1A). This sequence was found to be different to that 282 \npredicted previously, highlighting the importance of having a complete genome sequence available 283 \nfor clinical reference isolates.  284 \nAnother characteristic of lineage B isolates and BAL062 is the presence of a KL58 sequence 285 \nat the CPS biosynthesis K locus, which was found to be widely distributed and present in both 286 \nclinical and environmental isolates. Granted the importance of the CPS and the influence of its 287 \nspecific structure on both virulence (40) and the application of alternate therapies such as 288 \nmonoclonal antibodies (41) and bacteriophage (42, 43), we used BAL062 to determine the K58-289 \ntype structure. The non-2-ulosonic acid component of the K-unit was found to be 8ePse5Ac7Ac, the 290 \n8-epimer of Pse5Ac7Ac. This sugar was only recently discovered in the A. baumannii K135-type 291 \nCPS (35), and it was later proposed that the genes responsible for conversion of Pse5Ac7Ac to 292 \n8ePse5Ac7Ac are located outside the K locus (44). Further work will be needed to identify the 293 \ngenetic determinant(s) for 8ePse5Ac7Ac for the K58 and K135 CPS forms. Nonetheless, the 294 \ncomposition and overall topology of the CPS produced by BAL062 was found to be related to the 295 \nK2 and K93 CPS as expected.  296 \nThe structure correlated with the genetic annotation of KL58 gene cluster using agreed 297 \nnomenclature is critical to future understanding of its role in or contribution to different phenotypes. 298 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 13 \nIn fact, a previous study that used the BAL062 TraDis library to identify genes involved in 299 \nsusceptibility to or tolerance of colistin (8) showed that that genes at the K locus as well as genes at 300 \nthe OC locus play a role. However, these genes were not identified as being in these locations. 301 \nHence, the role of genes involved in synthesis of the outer core of LOS was not noticed. The 302 \npotential role of CPS in colistin resistance or the involvement of K locus genes in synthesis of the 303 \nLOS was also neither noticed nor explained, and further work will be needed to explain the role of 304 \nthe genes in colistin resistance. However, the location of genes in the K and OC loci was correctly 305 \nidentified in later studies (10, 11).  306 \n 307 \nMATERIALS AND METHODS 308 \nBacterial strain and antibiotic resistance profiling 309 \nA. baumannii isolate BAL062 was recovered in 2009 from a patient with ventilator associated 310 \npneumonia who was admitted to the ICU of the HTD in Ho Chi Minh City, Vietnam (6). The 311 \nantibiotic resistance profile of BAL062 was determined as described previously (45). 312 \n 313 \nBioinformatics analysis 314 \nThe complete genome of BAL062 was downloaded from NCBI assembly accession number 315 \nGCA_900088705.1 (chromosome: LT594095.1; plasmid: LT594096.1). KL and OCL sequences 316 \nwere identified by command-line Kaptive v 2.0.7 using the current A. baumannii KL (28) and OCL 317 \n(30) reference sequence databases. BLASTn was used to search 22,218 A. baumannii genomes in 318 \nthe NCBI GenBank and non-redundant databases (available as of 7th February, 2024) for further 319 \ninstances of the KL58 sequence, and the associated metadata (country, collection year and source of 320 \nisolation) were extracted from corresponding NCBI records and are compiled in Supplementary 321 \nTable S1. For isolates reported in Schultz et al., draft genome sequences were assembled from short 322 \nread data (SRA accessions listed in Table S1) using SPAdes (46). 323 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 14 \nMultilocus sequence typing (MLST) was performed using the A. baumannii Institut Pasteur 324 \nscheme available at (https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_seqdef). ResFinder v 325 \n4.4.2 (47) was used to detect antibiotic resistance genes. The core-SNP maximum likelihood 326 \nphylogeny was constructed using the Bactmap pipeline (https://github.com/nf-core/bactmap) with 327 \nrecombination removed using Gubbins (48), and the tree was visualised using iTOL 328 \n(https://itol.embl.de/). Figures were created using EasyFig v 2.2.2 (49) and annotated in Adobe 329 \nIllustrator. 330 \n 331 \nIsolation of capsular polysaccharide 332 \nBAL062 was cultivated in 2×TY media overnight. Bacterial cells were harvested by centrifugation 333 \n(10,000×g, 15 min), washed with and suspended in phosphate buffered saline. The suspension was 334 \ncooled down to 4 °C, 0.2 volume of CCl3CO2H was added, cells were precipitated by centrifugation 335 \n(15,000×g, 20 min), and two volumes of acetone were added to the supernatant. After intense 336 \nshaking, a crude CPS preparation was separated by centrifugation (8,000×g, 20 min), dissolved in 337 \nwater, the pH value was adjusted to pH 8 by adding 1 M NaOH, the CPS was precipitated with 338 \nacetone and separated by centrifugation as above, dissolved in distilled water and applied to a 339 \ncolumn (53 × 3.5 cm) of Sephadex G-50 Superfine (Healthcare). Elution was performed with 0.1% 340 \nHOAc and monitored using a UV-detector (Uvicord, Sweden) at 206 nm to give purified CPS 341 \nsamples. 342 \n 343 \nMonosaccharide analysis  344 \nCPS samples (1 mg) were hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were 345 \nconverted conventionally into the alditol acetates analyzed by GLC on a Maestro (Agilent 7820) 346 \nchromatograph (Interlab, Russia) equipped with an HP-5 column (0.32 mm  30 m) using a 347 \ntemperature program of 160 C (1 min) to 290 C at 7 C min-1. 348 \n 349 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 15 \nSmith degradation 350 \nA CPS sample (54 mg) from A. baumannii BAL062 was oxidized with aqueous 0.05 M NaIO4 (1 351 \nmL) 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 \nNaBH4 was destroyed with concentrated AcOH, the solution was evaporated, and the residue was 353 \nevaporated with methanol (3  1 mL), dissolved in water (in 0.5 mL) and applied to a column (35  354 \n2 cm) of TSK HW-40. The degraded polysaccharide was eluted with aqueous 0.1% AcOH and 355 \nhydrolyzed with 2 % HOAc (100 °C, 2 h) to give the -8ePseAc2-(2→1)-Gro glycoside (5.2 mg) 356 \nand a linear GlcNAc polymer (main-chain polysaccharide, 12 mg), which were isolated by gel-357 \npermeation chromatography on a column (108  1.2 cm) of TSK HW-40 in 1% HOAc.  358 \n 359 \nNMR spectroscopy  360 \nSamples were deuterium-exchanged by freeze-drying from 99.9 % D2O and then examined as 361 \nsolutions in 99.95 % D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz 362 \nspectrometer (Germany) at 60 °C. Sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (H 0, C 1.6) was 363 \nused as internal reference for calibration. 2D NMR spectra were obtained using standard Bruker 364 \nsoftware, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. 60-ms 365 \nMLEV-17 spin-lock time and 150-ms mixing time were used in 1H,1H TOCSY and ROESY 366 \nexperiments, respectively. A 60-ms delay was used for evolution of long-range couplings to 367 \noptimize 1H,13C HMBC experiments for the coupling constant of JH,C 8 Hz. 1H and 13C chemical 368 \nshifts were assigned using two-dimensional 1H,1H COSY, 1H,1H TOCSY, and 1H,13C HSQC 369 \nexperiments (Table 2). 370 \n  371 \nAcknowledgements 372 \nWe thank A/Prof Amy Cain (Macquarie University, Australia) for providing A. baumannii isolate 373 \nBAL062, and Dr Stephanie Ambrose (University of Sydney, Australia) for technical assistance. 374 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 16 \nNMR spectra were recorded in the Department of Structural Studies of N.D. Zelinsky Institute of 375 \nOrganic Chemistry, Moscow.  376 \n 377 \nFunding 378 \nThis work was supported by the Russian Science Foundation (grant number 19-14-00273), an 379 \nAustralian Research Council (ARC) Future Fellowship (FT230100400) to JJK, and a National 380 \nHealth and Medical Research Council (NHMRC) Investigator grant (GNT1194978) to RMH. 381 \n 382 \nReferences  383 \n1.  Murray CJL, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, Han C, Bisignano 384 \nC, Rao P, Wool E, Johnson SC. 2022. Global burden of bacterial antimicrobial resistance in 385 \n2019: a systematic analysis. The Lancet 399(10326):629-655. 386 \n2.   Isler B, Doi Y, Bonomo RA, Paterson DL. 2019. New treatment options against 387 \ncarbapenem-resistant Acinetobacter baumannii infections. Antimicrob Ag Chemother 388 \n63(1):e01110-18. 389 \n3.   Jacobs AC, Thompson MG, Black CC, Kessler JL, Clark LP, McQueary CN, Gancz HY, 390 \nCorey BW, Moon JK, Si Y, Owen MT. 2014. 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Rapid phylogenetic analysis of large samples of recombinant bacterial whole 549 \ngenome sequences using Gubbins. Nucleic Acids Res 43(3):e15. 550 \n49. Sullivan MJ, Petty NK, Beatson SA. 2011. Easyfig: a genome comparison visualizer. 551 \nBioinformatics 27(1):1009-1010. 552 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 23 \nFigure Legends 553 \nFigure 1. (A) Tn2008VAR in the BAL062 chromosome (base positions 3893121-3898596). 554 \nThe locus tags indicated on either side are the remnants of the interrupted acyl-CoA 555 \ndehydrogenase gene in the chromosome. The sequence of the 9 bp target site duplication is 556 \nshown next to the flags. (B) Genetic arrangement of AbGRIs in BAL062 chromosome: 557 \nAbGRI1 (base positions 3779179-3801151); AbGRI2 (base positions 2675982-2686299); 558 \nAbGRI3 (base positions 1400803-1408181). Green boxes indicate insertion sequences, red 559 \nare resistance genes, orange box is CR2, and flanking chromosomal genes are black. (C) 560 \nCore-SNP maximum likelihood phylogeny of GC2 genomes carrying KL58 from Vietnam 561 \nHTB outbreak reported in Schultz et al. (SRA accession numbers listed in Table S1). 562 \nBAL062 is shown in red. Year of collection, isolation source, STIP and OCL for each isolate 563 \nare shown next to a presence/absence matrix of antibiotic resistance genes coloured by class. 564 \nLineages indicated in Schultz et al. are indicated.  565 \n 566 \nFigure 2. Comparison of KL58 in the BAL062 chromosome (base positions 3972158-567 \n3948095) with KL2 from A. baumannii A74 (GenBank accession number KJ459911) and 568 \nKL93 from A. baumannii B11911 (GenBank accession number CP021345.1; bases 3338181-569 \n3368604). Genes coloured by function of gene product and grey shading is tBLASTx 570 \nidentity. Colour scheme and scale shown below. 571 \n 572 \nFigure 3. (A) 13C NMR spectra of the CPS of A. baumannii BAL062. (B) Parts of a two-573 \ndimensional 1H,13C HSQC spectrum of the CPS of A. baumannii BAL062. The 574 \ncorresponding parts of the one-dimensional 1H and 13C NMR spectra are displayed along the 575 \naxes. For designations of the monosaccharide residues see Figure 4 and Table 2.  576 \n 577 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 24 \n 578 \nFigure 4. (A) Structure of the CPS produced by A. baumannii BAL062 compared with K2 579 \n(32, 33) and K93 (34). (B) Products derived by chemical cleavages of the BAL062 CPS. 580 \n8ePse5Ac7Ac indicates 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-581 \nulosonic acid (di-N-acetyl-8-epipseudaminic acid); Gro indicates glycerol. 582 \n 583 \nFigure 5. Distribution of KL58 in A. baumannii genome sequences. Colour scheme 584 \ndenoting STs in the Institut Pasteur scheme is shown below. NCBI accession numbers for 585 \nisolates carrying KL58 are listed in Table S1. 586 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 25 \nTables 587 \nTable 1. Updated gene annotations for the KL58 and OCL1 loci in the BAL062 genome 588 \nGene \nname \nLocus tag GenPept \naccession \nAnnotation in \nLT594095.1  \nFunction/Predicted Function \nKL58 locus \nwzc BAL062_03872 SBS23916.1 ptk Protein tyrosine kinase \nwzb BAL062_03871 SBS23915.1 ptp Low molecular weight protein tyrosine \nphosphatase \nwza BAL062_03870 SBS23914.1  Outer membrane protein \ngna BAL062_03869 SBS23913.1 tuaD_2 UDP-N-acetyl-galactosamine dehydrogenase \npsaA BAL062_03868 SBS23912.1 capD UDP-N-acetylglucosamine 4,6-dehydratase/5-\nepimerase \npsaB BAL062_03867 SBS23911.1 arnB C4-aminotransferase \npsaC BAL062_03866 SBS23910.1 neuA Cytidylyltransferase \npsaD BAL062_03865 SBS23909.1  Nucleotidase \npsaE BAL062_03864 SBS23908.1  N-acetyltransferase \npsaF BAL062_03863 SBS23907.1 spsE Condensase \nwzx BAL062_03862 SBS23906.1  Oligosaccharide-unit translocase \ngtr118 BAL062_03861 SBS23905.1 lst Glycosyltransferase \nwzy BAL062_03860 SBS23904.1  Oligosaccharide-unit polymerase \ngtr8 BAL062_03859 SBS23903.1 tagE Glycosyltransferase \ngtr9 BAL062_03858 SBS23902.1 lsgF Glycosyltransferase \nitrA2 BAL062_03857 SBS23901.1 wcaJ GalNAc-1P initiating transferase \ngalU BAL062_03856 SBS23900.1 galU UDP-glucose-1-phosphate uridylyltransferase \nugd BAL062_03855 SBS23899.1 tuaD_1 UDP-glucose 6-dehydrogenase \ngpi BAL062_03854 SBS23898.1 pgi glucose-6-phosphate isomerase \ngne1 BAL062_03853 SBS23897.1 galE_2 UDP-glucose/UDP-N-acetyl-glucosamine 4-\nepimerase \natr42 BAL062_03852 SBS23896.1  Acetyltransferase \natr43 BAL062_03851 SBS23895.1  Acetyltransferase \npgm BAL062_03850 SBS23894.1 manB Phosphoglucomutase/phosphomannomutase \nOCL1 locus \ngtrOC1 BAL062_00583 SBS20708.1  Glycosyltransferase \ngtrOC2 BAL062_00584 SBS20709.1  Glycosyltransferase \npda1 BAL062_00585 SBS20710.1 icaB Polysaccharide deacetylase \ngtrOC3 BAL062_00586 SBS20711.1 lpsC Glycosyltransferase \ngtrOC4 BAL062_00587 SBS20712.1  Glycosyltransferase \norf1 \n(ghy) \nBAL062_00588 SBS20713.1  Unknown \ngtrOC5 BAL062_00589 SBS20714.1  Glycosyltransferase \ngtrOC6 BAL062_00590 SBS20715.1  Glycosyltransferase \ngtrOC7 BAL062_00592 SBS20717.1 sacB Glycosyltransferase \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n 26 \nTable 2. 1H and 13C NMR chemical shifts (δ, ppm) of the capsular polysaccharide produced 589 \nby A. baumannii BAL062. 590 \nSugar С3 or C1 \nH3ax,H3eq \nor H1 \nС4 or C2 \nH4 or H2 \nС5 or C3 \nH5 or H3 \nС6 or C4 \nH6 or H4 \n \nС7 or C5 \nH7 or H5 \nC8 or C6 \nH8 or H6 \nС9 \nH9 \n-8ePseAc2 D 37.5 \n1.62, 2.50 \n67.8 \n3.90 \n49.8 \n4.18 \n73.3 \n3.87 \n55.0 \n3.89 \n67.5 \n4.37 \n19.2 \n1.08 \n-6)--Gal C 100.0 \n4.96 \n70.2 \n3.83 \n71.2 \n3.97 \n71.3 \n3.83 \n71.2 \n3.99 \n65.7 \n3.57, 3.97 \n \n-3,6)--Glc B 106.0 \n4.78 \n74.7 \n4.04 \n85.8 \n3.91 \n70.0 \n4.10 \n75.6 \n3.68 \n67.8 \n3.74, 3.80 \n \n-3)--GalNAc A 103.4 \n4.51 \n53.5 \n3.40 \n81.5 \n3.66 \n70.0 \n3.59 \n76.7 \n3.62 \n62.9 \n3.74, 3.95 \n \n1H NMR chemical shifts are italicized. 591 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\nBAL225\nBAL230\nBAL315\nBAL219\nBAL215\nBAL295\nBAL238\nBAL298\nBAL128\nBAL383\nBAL372\nBAL369\nBAL377\nBAL350\nBAL341\nBAL346\n354n\nBAL339\nBAL084\nBAL114\nBAL056\nBAL064\nBAL062\nUV1897\nTree scale: 0.0001\nLineage B\nLineage A\nLineage E\nLineage C\nC.\n2006\n2007\nAminoglycosides\n3rd GenerationCephalosporins\nblaTEM-1D\nblaPER-1\noxa23\ntet(B)\ntet39\nmphE\nmsrE\nsul2\nsul1\ncatB8\nfloR\narmA\naphA1\naacC1\naadA1\naadA24\nstrA/strB\nCarbapenems\nTetracyclines\nMacrolides\nSulfonamides\nPhenicols\nRifamycin\nYear\nSource\nCarriage\nVAP\nST IP\naacA4\naadB\narr2\naphA6\naac(3)-IId\noxa58\n2009 VAP\n2009 VAP\n2009 VAP\n2009 VAP\n2009 VAP\n2011 VAP\n2011 VAP\n2011 VAP\n2011 VAP\n2012 VAP\n2012 VAP\n2012 VAP\n2010 VAP\n2011 VAP\n2010 VAP\n2011 VAP\n2010\n2010\nVAP\nVAP\n2011 VAP\n2010 VAP\n2010 VAP\nIsolate\n2\n1550\n2\n2\n2\n2\n571\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2\n2012\nVAP\nOCL\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n18\n1\n1\n1\n1\n1\n1\n1\n1\nTn2008VAR\nA.\nAbGRI2\nAbGRI3\nΔcomMtniC tniA tniB tniD tniE orf uspA sup orf4 tetR(B)tetA(B) rcr2 strB strA orf4b\nAbGRI1\norf IS26 orfΔ orf orf\nB. ΔcomM\n     (CACCGATCC)\nBAL062_03807 BAL062_03800\nISAba33 ISAba1oxa23\n     (CACCGATCC)     \n     (CTCATCCT)\norf asr\nIS26 IS26tnpAΔtnpRtnpMintI1aacC1PaadA1sul1orf5 PQIS6100Δ qacEdelta1\n     (CGCCAACT)\nΔ10,253 bp\nΔ1,156 bp\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\nThe K locus (KL)\nCMP-Pse5Ac7Ac synthesisCPS export Simple sugar synthesis\nKL58\ngtr9gtr8 atr42wzc wzb wza gna psaDpsaEpsaA psaB psaC psaF wzx wzyKL58gtr118 itrA2 galU gpi gne1ugd pgmatr43\nREGION 1 REGION 2 REGION 3\ngtr5gtr4wzc wzb wza gna psaDpsaEpsaA psaB psaC psaF wzx wzyKL2kpsS1 itrA2 galU gpi gne1ugd pgmgtr3\nKL2\n1 kb\nCapsule export machinery\nNucleotide-sugar biosynthesis Acetyl or acyl transferase (atr)\nRepeat unit processing\nGlycosyltransferase (gtr)\nInitiating transferase (itr)\nSimple sugar synthesis Other \n60%\n100%\ntBLASTx % identity\ngtr5gtr25wzc wzb wza gna psaG psaHpsaA psaB psaC psaF wzx wzyKL93gtr167 itrA2 galU gpi gne1ugd pgmpgt\nKL93\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\nA.\nB.\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\n→3)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→\nWzyKL58\n[ItrA2]\nGtr9\n   6)\nGtr118    (1\nGtr8\n→\nCPS\nBAL062 \nWzyKL58\n α-8ePse5Ac7Ac-(2→6)-α-D-Galp\n→3)-β-D-Galp-(1→3)-β-D-GalpNAc-(1→\n[ItrA2]\nGtr5\nGtr4\n   6)\n α-Pse5Ac7Ac-(2→6)-β-D-Glcp\n   (1\n→KpsS1\nK2\n→3)-β-D-Galp-(1→3)-β-D-GalpNAc-(1→\n[ItrA2]   6)\nGtr5\nβ-Pse5Ac7R-(2→6)-α-D-Galp\n   (1\nGtr25\n→Gtr167\nK93\nWzyKL2WzyKL2\nWzyKL93WzyKL93\nAB\nCD\n→3)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→\nMainchain polysaccharide\nAB\n8ePse5Ac7Ac-(2→1)-Gro\nD\nGlycoside 1\nα-8ePse5Ac7Ac-(2→6)-α-D-Galp-(1→6)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→\nTetrasaccharide 2\nABCD\nA. B.\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint \n\nIHIT35900\nPUMA0184\nPUMA0214\n98_E23.3\n16-Klo_64-1\nPUMA0123\nLWSM-0248\nWU_MDCI_Ab184\n17-Lo_4-1\n29D2\n2023EL-00143\n2023EL-00144\n2022JQ-00544\n2021CK-01311\nMRSN1339\nMRSN31468\nMRSN7782\nAb-69\nAB179_VUB\nSH37\n350_n\n344_an\n341_c\n316_an\n277_ax\nUV_1268\nBAL383\nBAL372\nBAL369\nBAL339\nBAL350\nBAL346\nBAL341\nBAL315\nBAL298\nBAL295\nBAL238\nBAL230\nBAL225\nBAL219\nBAL215\nBAL128\nBAL114\nBAL084\nBAL064\nBAL062\nBAL056\nUV_1897\n354_n\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nChina\nBelgium\nChina\nUSA\nGermany\nUSA\nUSA\nUSA\nUSA\nUSA\nPoland\nGermany\nUSA\nGermany\nSingapore\nPoland\nGermany\nSingapore\nSingapore\nUSA\n2010\n2006\n2007\n2009\n2009\n2009\n2009\n2009\n2010\n2010\n2010\n2010\n2010\n2011\n2011\n2011\n2011\n2011\n2011\n2011\n2012\n2012\n2012\n2005\n2005\n2006\n2006\n2006\n2006\n2010\n2017\n2020\n2006\n2003\n2010\n2021\n2022\n2023\n2023\n2014\n2017\n2018\n2019\n2023\n2016\n2019\n2023\n2023\n2014\nblaTEM-1D\nblaPER-1\nblaCARB-1\noxa23\ntet(B)\ntet39\nmphE\nmsrE\nsul2\nsul1\ncatB8\nfloR\narmA\naphA1\naacC1\naadA1\naadA24\nstrA/strB\nStrain\nCountry\nYear\nAb11\nAb9\nAb14\nPUMA0140\nWU_MDCI_Ab140\nMST-SNC-9\nPUMA0145\nAb182\nPUMA0099 Singapore\nUSA\nSingapore\nCanada\nUSA\nSingapore\nNA\nNA\nNA\n2023\n2018\n2023\nNA\n2018\n2023\nNA\nNA\nNA\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nVietnam\nSource\nCarriage\nConnective \ntissue infection\nAgricultural \nsurface water\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nVAP\nCarriage\nCarriage\nCarriage\nCarriage\nCarriage\nNA\nSputum\nUrine\nWound\nGroin\nSurveillance\nRectal swab\nBlood\nBlood\nCatheter tip\nWhite stork\nNestling\nEarthworm\nRespiratory \ntract\nPig production \nsetting\nNA\nWhite stork\nBoot swab\nNA\nNA\nLizard faeces\nNA\nUrine\nNA\nNA\nNA\nNA\nNA\nST IP\nST2 ST1550\nST571 ST574\nST10 ST150\nST154\nST309\nST858\nST342\nST1220\nST1301\nST2373\nST2561\nST2643\nNon-typeable\naacA4\naadB\narr2\naphA6\naac(3)-IId\noxa58\ntet(X6)\naph(4)-Ia\naac(3)-IV\nOCL\n1\n1\n1\n1\n6\n6\n6\n6\n2\n1\n1\n1\n2\n1\n1\n2\n2\n2\n2\n2\n2\n1\n1\n1\n1\n1\n1\n18\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n6\n6\n6\n1\n1\n1\n1\n1\n1\n1\n2\n3\n1\nGC2/CC2 CC10 Other\nBAL377 2012Vietnam VAP 1\nAminoglycosides\n3rd GenerationCephalosporins\nCarbapenems\nTetracyclines\nMacrolides\nSulfonamides\nPhenicols\nRifamycin\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted May 9, 2024. ; https://doi.org/10.1101/2024.05.09.593323doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}