Evolution of carbapenem-resistant Acinetobacter baumannii through whole 1

genome sequencing and comparative genomic analysis 2

3

Henan Lia#

, Fei Liub#

, Yawei Zhanga, Xiaojuan Wang

a, Chunjiang Zhao

a, Hongbin 4

Chena, Feifei Zhang

a, Baoli Zhu

b, Yongfei Hu

b*, Hui Wang

a* 5

6

7

Department of Clinical Laboratory, Peking University People’s Hospital, Beijing, 8

Chinaa; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of 9

Microbiology, Chinese Academy of Sciences, Beijing, Chinab 10

11

Running title: Evolution of carbapenem-resistant A. baumannii 12

Keywords: Acinetobacter baumannii; comparative genomics; antibiotic resistance; 13

evolution. 14

15

# Henan Li and Fei Liu contributed equally to this work. 16

*Corresponding authors: 17

Prof. Hui Wang, Department of Clinical Laboratory, Peking University People’s 18

Hospital, Beijing, 100044, China, Tel. +86-10-88326300; Email: whuibj@163.com 19

(H. Wang). 20

Yongfei Hu, Key Laboratory of Pathogenic Microbiology and Immunology, Institute 21

of Microbiology, Chinese Academy of Sciences, Beijing, China, Tel. 22

+86-10-64807433; Email: huyf@im.ac.cn (Y. Hu). 23

24

AAC Accepts, published online ahead of print on 8 December 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.04609-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

ABSTRACT Acinetobacter baumannii is a globally important nosocomial pathogen 25

characterized by an evolving multidrug resistance. A total of 35 representative clinical 26

A. baumannii strains isolated from 13 hospitals in nine cities of China during 27

1999–2011, including 32 carbapenem-resistant and three susceptible A. baumannii, 28

were selected for whole genome sequencing and comparative genomic analysis. 29

Phylogenetic analysis revealed that the earliest strain 1999BJAB11 and two Zhejiang 30

strains isolated in 2004 were the founder strains of carbapenem-resistant A. baumannii. 31

Ten types of resistance abaR islands were identified, and a previously unreported 32

abaR island was identified in two Zhejiang strains isolated in 2004, which comprised 33

two-component response regulator, resistance-related proteins, and RND efflux 34

system proteins. Multiple transposons or insertion sequences (ISs) existed in each 35

strain, which gradually tended to diversify with evolution. Some of these IS elements 36

or transposons were the first to be reported, and most of them were mainly found in 37

strains from two provinces. Genome feature analysis illustrated diversified resistance 38

genes, surface polysaccharides, and restriction-modification system, even in strains 39

that were phylogenetically and epidemiologically very closely related. IS-mediated 40

deletions were identified in the T6SS region, csuE region, and core 41

lipooligosaccharide (LOS) loci. Recombination occurred in the heme utilization 42

region and intrinsic resistance genes blaADC and blaOXA-51-like variants, and three novel 43

blaOXA-51-like variants, blaOXA-424, blaOXA-425, and blaOXA-426, were identified. Our results 44

could improve the understanding of the evolutionary processes that contribute to the 45

emergence of carbapenem-resistant A. baumannii, and help in elucidating the 46

molecular evolutionary mechanism in A. baumannii. 47

48

49

Acinetobacter baumannii is an important opportunistic pathogen that has caused 50

severe nosocomial infections worldwide(1). Multidrug-resistant A. baumannii, 51

resistant to carbapenems, has been increasingly reported worldwide, which raises 52

serious concerns about the limited antimicrobial treatment options available (2). Our 53

previous study indicated that the percentage of imipenem- and meropenem-resistant A. 54

baumannii had increased from 4.5% to 61.7% and 4.5% to 62.8% during 2003–2010 55

in China. In 2012, according to the Chinese Meropenem Surveillance Study (CMSS), 56

the susceptibility rates of A. baumannii to imipenem and meropenem were 37.8% and 57

36.0%, respectively (3). 58

A. baumannii rapidly develops multidrug resistance due to the presence of mobile 59

genetic elements, such as insertion sequences (ISs), transposons, and resistance 60

islands (4). Many recent studies have highlighted the diversity in the genomic location, 61

architecture, and content of resistance islands, demonstrating the dynamic nature of A. 62

baumannii antibiotic resistance mechanisms and the adaptive significance of these 63

elements (5-7). However, evolution of antibiotic resistance over time in relation to 64

changes in the major clones in China has not yet been investigated. Moreover, only a 65

few studies have focused on the genetic background differences among A. baumannii 66

isolates collected from different locations at different time periods. 67

Comparative genomics study helps in the evaluation of the resistance 68

mechanisms, pathogenicity, and evolution of bacterial pathogens at genome-wide 69

level (8). Despite increased research on A. baumannii epidemiology and evolution 70

(9-12), large gaps remain in our understanding of the evolutionary processes that 71

contribute to multidrug resistance and genomic diversification of A. baumannii, 72

especially in China. In the present study, a total of 35 representative A. baumannii 73

strains isolated from 13 hospitals in 9 cities of China during 1999–2011 were selected 74

for comparative whole genome sequencing to examine strain-level genetic diversity 75

and gene content variation. Through the analysis of these strains with different 76

backgrounds, we aimed to gain a better understanding of the resistance evolution of A. 77

baumannii in China.78

MATERIALS AND METHODS 79

Strain isolation and genotypic and phenotypic characterization. The A. baumannii 80

strains were collected from 13 hospitals in nine cities of China from 1999 to 2011. 81

The isolates from 1999 to 2005 were selected from 221 carbapenem-resistant A. 82

baumannii (CRAB) in our previous study (13), and the isolates from 2011 were 83

selected from 283 A. baumannii strains in the Chinese Antimicrobial Resistance 84

Surveillance of Nosocomial infections (CARES) (14). The isolates were subjected to 85

additional analysis to characterize the antibiotic phenotypes (Table S1) and genotypes 86

via MLST (http://pubmlst.org/abaumannii/) (15). From this collection of isolates, 35 87

representative isolates were selected from predominant clone in different locations, 88

dates, and sources. To put the sequenced strains in a phylogenetic context and assess 89

the shared and clade-specific gene contents, the genomes were compared with 17 90

reference Acinetobacter genomes available in NCBI as of January 2014. 91

DNA preparation, library construction, sequencing, and assembly. DNA was 92

isolated with the DNA purification kit (Qiagen). Illumina sequencing libraries were 93

prepared by using Nextera kits with indexed-encoded adapters from Illumina, 94

according to the manufacturer’s instructions. The libraries were pooled for sequencing 95

on MiSeq, and paired-end sequence reads were obtained representing 50–200-fold 96

genome coverage. The Illumina sequence data were assembled using SOAP denovo 97

(Version 2.04). PCR amplification and Sanger sequencing were used to solve the 98

ambiguity of the order and orientation of scaffolds. 99

Genome annotation. The assembled genome sequence was annotated by the 100

NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), using Glimmer 101

3.0 for the identification of protein-coding genes (16), tRNAscan-SE for tRNA genes 102

(17), and RNAmmer for rRNA genes (18). The ISs were identified using the IS Finder 103

database (www-is.biotoul.fr) (19). The origin of replication (oriC) and putative DnaA 104

boxes were identified using Ori-Finder (20). 105

Whole genome phylogeny construction. Multiple sequence alignments of 52 106

genomes were performed using Mugsy (21).The tree was constructed based on SNPs 107

from the whole genome alignment. This alignment included SNPs from 17 reference 108

genomes and 35 genomes sequenced in this study. 109

eBURST analysis. eBURST analysis was employed to investigate the 110

evolutionary relationships and CCs within the isolates, using the software on the 111

eBURST website (http://eburst.mlst.net/v3/enter_data/single/), with statistical support 112

for the complexes being assessed via the bootstrap resampling method with 1000 113

resamplings (22). The analysis was performed using both stringent (a minimum of six 114

shared alleles) and relaxed (a minimum of five shared alleles) grouping parameters. 115

Estimation of core and pan-genome size. A BLASTP search between every pair 116

of protein sequences from each strain was performed. PanOCT was used to identify 117

the orthologs with the BLASTP output (23). 118

Nucleotide sequence accession numbers. This Whole Genome Shotgun project 119

has been deposited at DDBJ/EMBL/GenBank under the accession PRJNA261018. 120

The three novel blaOXA types were designated as blaOXA-424 (GenBank accession 121

number: KM588352), blaOXA-425 (GenBank accession number: KM588353), and 122

blaOXA-426 (GenBank accession number: KM588354) (http://www.lahey.org/Studies/). 123

124

RESULTS 125

A total of 35 representative A. baumannii, including 32 carbapenem-resistant A. 126

baumannii (CRAB) and three susceptible A. baumannii, isolated from patients with 127

hospital-acquired infections in 13 hospitals in nine cities of China during 1999–2011 128

were selected for whole genome sequencing. The 32 CRAB isolates consisted of one 129

isolate from 1999, eight isolates from 2003–2005, and 23 isolates from 2011. Most of 130

the isolates were collected in Beijing, China (14/35, 40%). The sources of these 35 131

isolates were sputum (20/35, 57.1%), blood (10/35, 28.6%), and abdominal fluid 132

(5/35, 14.3%). The general characteristics of the 35 A. baumannii genomes are listed 133

in Table S2. 134

Whole genome phylogeny of the genus Acinetobacter. To facilitate detailed 135

analysis of strain relationships, we developed a phylogeny based on single nucleotide 136

polymorphisms (SNPs) from whole genome alignment to represent the ancestral 137

relationships among 35 strains and 16 other A. baumannii strains (Fig. 1A). These 138

included 12 multidrug-resistant A. baumannii strains (AYE, AB0057, ACICU, 139

AB1656-2, TYTH-1, TCDC-AB0715, BJAB07104, BJAB0715, BJAB0868, ZW85-1, 140

MDR-TJ, and MDR-ZJ06), two susceptible strains (ATCC17978 and AB307-0294), 141

one community-acquired strain D1279779, and one non-clinical strain SDF isolated 142

from a human body louse. ADP1, a soil-living Acinetobacter baylyi strain was used as 143

the outgroup for comparison. 144

Based on the phylogenetic data, all the strains, along with nine previously 145

reported Asian strains, including MDR-ZJ06, MDR-TJ, ZW85-1, BJAB07104, 146

BJAB0715, and BJAB0868 from mainland China, TCDC-AB0715 and TYTH-1 from 147

China, Taiwan, and AB1656-2 from Korea, were grouped together with ACICU, a 148

strain of global clone II (GC II) group. The susceptible strains were clustered on the 149

edge of the evolutionary tree. Two groups of founder strains were identified among 150

the CRAB based on the phylogenetic tree, which were the earliest isolated strain 151

1999BJAB11 and two strains from Zhejiang isolated in 2004. Interestingly, the 152

earliest isolated strain 1999BJAB11 was separated from all of the other ST92 strains, 153

suggesting that it may have a different genome content, when compared with the other 154

ST92 strains. 155

The eBURST algorithm revealed one clonal complex (CC) CC1 and six 156

singletons (Fig. 1B). The CC1 contained ST92, which was identified as a potential 157

founder, with ST75, ST137, ST90, ST118, and ST138 radiating from it. All the 158

isolates in CC1 were CRAB, and the three susceptible strains were clustered into 159

singletons. The location and source of these isolates showed diversity in different ST. 160

Evolution of the resistance abaR islands. Ten types of the resistance abaR 161

islands were identified in the 32 CRAB strains (Fig. 2A). Except for the type 6 abaR 162

island, other types shared high homology. Type 10 abaR island was the predominant 163

type, which was identified in 12 strains (12/32, 37.5%), and was 19.123 kb in length 164

and contained the highest number of resistance genes, including tniB, usp, sul, tetA, 165

tetR, arsR, and aph, similar to those noted in a strain from Japan with one different 166

nucleotide (24). Type 5 abaR island in 1999BJAB11 contained the largest number 167

genes, and the conjugal transfer gene traA was identified only in this isolate. The 168

resistance genes tetA, tetR, arsR, aphA, and strB were not found in type 1 abaR island. 169

While the sulfonamide resistant protein dihydropteroate synthase and 170

phosphoglucosamine mutase (glmM) were detected in type 2, 7, and 10 abaR islands. 171

The phosphoglucosamine mutase (glmM) can catalyze the conversion of 172

glucosamine-6-phosphate to glucosamine-1-phosphate, which is an essential step in 173

the formation of the cell wall precursor UDP-N-acetylglucosamine (25). In 174

Streptococcus gordonii, mutations in glmM appear to influence bacterial cell growth 175

and morphology, biofilm formation, and sensitivity to penicillins (26). Function of 176

glmM in A. baumannii was currently not well known. The mobile element protein was 177

not observed in type 3 and 10 abaR islands. Two strains from Zhejiang isolated in 178

2011 contained type 9 abaR island, which showed gene arrangement when compared 179

with type 8 abaR island. Two Zhejiang strains collected in 2004 had the type 6 abaR 180

island, which included the two-component response regulator, osmosensitive K+ 181

channel histidine kinase KdpD, methyl viologen resistance protein smvA, RND 182

multidrug efflux transporter acriflavin resistance protein, and RND efflux system 183

membrane fusion protein CmeA. These genes were not observed in the other nine 184

types of abaR islands. The evolutionary ideograph of the abaR islands is shown in Fig. 185

2B, which was according to the time. The type 5 abaR island in the earliest strain 186

1999BJAB11 contained the largest number genes, and with the genome evolution, 187

gene acquirement, gene loss and gene rearrangement were detected in the strains 188

isolated during 2003-2011. The type 6 abaR island in two Zhejiang strains was 189

relatively independent of the other strains during abaR islands evolution. 190

Variations in transposon-associated antibiotic resistance genes. The 191

transposons containing drug resistance genes varied in different A. baumannii strains 192

(Fig. 3A). BlaOXA-51-like gene was located in three types of transposons. Eleven strains 193

possessed IS-blaOXA-51-like-IS transposon, and 10 of them were flanked by two ISAba1 194

elements. One strain 2011ZJAB2 was flanked by ISAba1 and ISAba16, and eight 195

strains possessed ISAba1-blaOXA-51-like-metallo-beta-lactamase-ISAba1 transposons. 196

Only one strain 2011LNAB2 possessed ISAba1-blaOXA-51-like- 197

class-A-beta-lactamase-ISAba1 transposon. ISAba1-metallo-beta-lactamase-ISAba1 198

was detected in 11 stains, but in two strains from Zhejiang isolated in 2011, 199

metallo-beta-lactamase was flanked by ISAba14 and ISAba16. 200

ISAba1-class-A-beta-lactamase-ISAba1 was identified in 13 strains. In one strain 201

from Zhejiang isolated in 2004, ISAba20 and ISAba1 were detected on both the sides 202

of class A beta-lactamase; and in two strains from Guangdong isolated in 2011, 203

ISAba1 and ISAba13 were detected. In seven strains isolated in 2011, blaADC-30 was 204

located in ISAba1-blaADC-30-ISAba1, but in 2005LNAB4, blaADC-30 was flanked by 205

ISAba1 and ISAba18, and in two strains from Zhejiang isolated in 2011, blaADC-30 was 206

flanked by ISAba1 and ISAba14. Furthermore, in two strains from Zhejiang isolated 207

in 2004, ISAba1-ampC-ISAba1 was identified. 208

The blaOXA-23-containing resistance island was identified in all the 32 CRAB 209

strains (Fig. 3B). In most of the sequenced strains (30/32, 93.8%), blaOXA-23 was 210

located in a transposon, which showed 99% identity to Tn2009. Tn2009 was initially 211

identified on the chromosome of A. baumannii MDR-ZJ06 from China, with a length 212

of 8421 bp and flanked by two ISAba1 elements. Tn2009 was noted to carry the 213

blaOXA-23 gene, the truncated DEAD/DEAH box helicase gene, the ATPase gene, the 214

yeeC gene, and the yeeB gene. However, in two isolates from Guangzhou obtained in 215

2011, blaOXA-23 was found in transposon Tn2006, which was shorter than Tn2009. 216

Tn2006 contained the ATPase gene, the yeeA gene, and the yeeB gene, and was 217

flanked by two ISAba1 elements. 218

Pan-genome: Core and accessory genes. All the sequenced A. baumannii 219

strains contained a genetically highly homogeneous core genome that encodes 220

proteins involved in DNA replication, transcription, and translation as well as many 221

metabolic pathways. By using the best reciprocal BLAST matches, we identified 2830 222

core genes/proteins and 1206 accessory genes/proteins among all the 35 A. baumannii 223

isolates. Graphing of the numbers of core and pan-genome as a function of the 224

number of strains sequenced revealed that the slope for the core gene number was 225

approaching an asymptote, whereas the pan-genome continued to expand even after 226

compilation of 35 genomes (Fig. 4A). 227

Variation in intrinsic chromosomal resistance genes related to carbapenem 228

resistance. The phylogenetic distribution of different alleles of blaOXA-51-like and 229

blaADC showed evidence of recombination and mutation. The presence of an upstream 230

insertion element, ISAba1, suggested that the entire region was replaced by 231

homologous recombination. The blaADC gene was always associated with upstream 232

ISAba1 oriented to allow overexpression of the gene, except in three susceptible 233

strains (Fig. 4B). The sequences from 19 strains were identical to the 234

extended-spectrum blaADC-30 variant (blue), and three strains showed 99% identity to 235

blaADC-30 (dark blue). Five strains contained blaADC-25 (yellow), and one strain carried 236

blaADC-56 (purple). One susceptible strain 2011BJAB9 and three resistant strains 237

showed 99% identity to blaADC-53 (orange), while blaADC in 1999BJAB1 showed 99% 238

identity to blaADC-29. The sequences from the susceptible strains 2011GDAB2 and 239

2011TJAB1 showed 98% and 99% identity to blaADC-52 and blaADC-63, respectively. 240

The blaOXA-51-like variants were different in the resistant strains and susceptible 241

strains. The most common blaOXA-51-like variant in resistant strains was blaOXA-66, 242

which was detected in 27 resistant strains (27/35, 77.1%), and blaOXA-68 were detected 243

in three resistant strains from Zhejiang. One unusual variant blaOXA-234 was detected 244

in a resistant strain from Jiangsu in 2005. Furthermore, three novel variants were 245

identified, including blaOXA-425 in the resistant strain 2011BJAB7 and blaOXA-424 and 246

blaOXA-426 in the susceptible strains 2011GDAB2 and 2011BJAB9, respectively. In the 247

susceptible strain 2011TJAB1, blaOXA-69 was detected. In most CRAB strains (25/32, 248

78%), blaOXA-51-like variants were associated with upstream ISAba1, but no upstream 249

ISAba1 was detected in all three susceptible strains. 250

IS-mediated T6SS and csuE region deletions. There were multiple instances of 251

chromosomal gene loss that might have possibly been mediated by an IS adjacent to 252

the deletion (Fig. 4C). Two large deletions adjacent to ISAba1 elements have been 253

previously described (10), including a 40-kbp region encoding the entire type VI 254

secretion system (T6SS) and a ~20-kbp region of adhesion genes (csuE) involved in 255

aspartate metabolism. In the present study, three different types of T6SS region were 256

noted, and eight strains lacked this region. Among these eight strains, in five strains, 257

deletion was mediated by IS insertion. It is worth noting that except for ISAba1 258

insertion in three strains isolated in 2011, ISAba13 insertion was detected in two 259

strains from Guangdong isolated in 2011. Similarly, three different types of csuE 260

region were also noted, and 12 strains lacked this region. Among these 12 strains, 261

deletion was mediated by IS insertion in three strains and transposase insertion in two 262

strains, respectively. Except for ISAba1-mediated deletion in 2011BJAB7, this study 263

is the first to identify ISAba125 insertion in two strains from Zhejiang isolated in 264

2004 and transposase insertion in the csuE region in two strains from Guangdong 265

isolated in 2011. 266

Variation in surface polysaccharide synthesis and heme utilization region. 267

The 35 strains showed substantial variation in the content and organization of loci 268

involved in surface polysaccharide synthesis (Fig. 4D). The core lipooligosaccharide 269

(LOS) loci (outer core [OC] locus) (27) consisted of one predominant type (blue), 270

which was present in most of the strains (29/35, 82.9%); however, we also identified 271

several variations in the three susceptible strains and one resistant strain. In two 272

strains from Zhejiang isolated in 2004, ISAba20 insertions were detected. With regard 273

to capsular (K locus) loci, 12 variations were identified. One predominant type (blue) 274

existed in 10 strains (28.6%), which was most closelgy related to ACICU (99.5% 275

identity at the nucleotide level). 276

The heme utilization region, containing several genes involved in heme 277

utilization, was more common in strains collected in 2011. However, this region was 278

absent in 1999BJAB1 and most of the strains collected during 2003–2005, except for 279

two strains from Zhejiang isolated in 2004. Among the 23 strains collected in 2011, 280

the heme utilization region was noted in 18 strains, which was 96% identical to 281

ACICU (Fig. 4E). 282

Variation in the restriction-modification system. The restriction-modification 283

(RM) system is a major participant in the co-evolutionary interaction between mobile 284

genetic elements (MGEs) and their hosts through the regulation of horizontal gene 285

transfer (HGT). In the present study, six strains clustered with two strains from 286

Zhejiang isolated in 2004 contained more RM system genes (Fig. 4F). In the 287

susceptible strain 2011TJAB1 and two resistant strains from Zhejiang isolated in 2004, 288

we identified Type I RM system methylase subunit, restriction subunit, and specificity 289

subunit. Furthermore, strains 2011ZJAB1 and 2011BJAB3 only had Type I RM 290

system methylase subunit, and strain 2011ZJAB4 only had Type I RM system 291

restriction subunit. Type II RM system methylase subunit was detected in strain 292

1999BJAB11 and susceptible strain 2011TJAB1, whereas Type III RM system 293

methylation subunit was found in three Zhejiang strains and two Guangdong strains. 294

CRISPR-associated protein was only detected in one susceptible strain 2011BJAB9. 295

Variation in phage-related regions. Phage-related regions are another source of 296

variability contributing to the difference among A. baumannii strains. Two 297

predominant types of phage-related regions were located within the same 298

chromosomal location corresponding to 1.11–1.16 Mbp in ACICU (ACICU_00997 to 299

ACICU_01077) and 1.45–1.53 Mbp in TYTH-1 (M3Q_1334 to M3Q_1458) (Fig. 300

4G). One primary variant typified by the completed reference genome of TYTH-1 301

consisted of two probable phage insertion events flanked by phage integrases (blue), 302

whereas another variant possessed only the second of the two phage elements (green). 303

Two susceptible strains 2011TJAB1 and 2011BJAB9 and strain 2011ZJAB4 had 304

different phage-related regions, when compared with the two main variants, and all 305

these strains were susceptible to tigecycline. 306

307

DISCUSSION 308

Despite increased research on A. baumannii evolution, the development of multidrug 309

resistance and genomic diversification of A. baumannii is not yet clear, especially in 310

China. In the present study, we have reported the whole genome sequences and 311

comparative genomic analysis of 35 representative A. baumannii strains isolated 312

during 1999–2011 in 13 hospitals in nine cities in China, including 32 CRAB and 313

three susceptible A. baumannii with different genotypes and phenotypes. These 314

genomes represent a significant addition of genomic data from the genus and could 315

serve as a valuable resource for future genomic studies. 316

It has been indicated that the resistance abaR islands have evolved into diverse 317

types through multiple events of insertion, deletion, and recombination (6, 28). The 318

predominance of type 4 abaR resistance islands among the CRAB isolates have been 319

reported in South Korea (29) and Taiwan (30). However, the evolution of resistance 320

abaR islands has not yet been investigated, especially in China. In the present study, it 321

was found that the type 6 abaR island in two Zhejiang strains collected in 2004 is 322

different from the previously identified abaR islands, which may thus be a novel abaR 323

island. The type 6 abaR island was noted to comprise the two-component response 324

regulator, osmosensitive K+ channel histidine kinase KdpD, methyl viologen 325

resistance protein smvA, RND multidrug efflux transporter acriflavin resistance 326

protein, and RND efflux system membrane fusion protein CmeA. These regulators 327

and resistance-related proteins in the type 6 abaR island may enhance the strain’s 328

adaptability and antibiotic resistance, which should be further studied. 329

Frequently encountered transposons and ISs carrying resistance genes in bacteria 330

reflect recombination flexibility during acquisition of resistance (4). In the present 331

study, each strain contained multiple transposons or ISs, which gradually tended to 332

diversify in the process of evolution. It must be noted that some of these ISs or 333

transposons are novel, and that most of the novel IS element insertion or transposons 334

were mainly found in strains from Zhejiang or Guangdong province of China. The 335

resistance genes or elements may spread through a transposon- or IS-mediated 336

mechanism, and the diversification of transposons or ISs in the process of evolution 337

increases the possibility of acquisition of novel resistance genes or elements. 338

Recombination has been reported to contribute to the change in the A. baumannii 339

genome (28). Furthermore, the heme utilization region is also highly variable among 340

the A. baumannii strains, which was more common in strains collected in 2011 in the 341

present study, thus making it difficult to assess whether this region was present in a 342

common ancestor and lost by some strains or was gained and subsequently transferred 343

via recombination to different lineages as initially observed. In addition, evidence of 344

recombination events in the allelic distribution of blaADC and blaOXA-51-like variants 345

was also noted. In all CRAB strains, the blaADC gene was associated with upstream 346

ISAba1 oriented to allow overexpression of the blaADC genes. However, in all three 347

susceptible strains, no upstream ISAba1 was detected. In addition, the blaOXA-51-like 348

variants were different in the resistant strains and susceptible strains. Four uncommon 349

blaOXA-51-like genes were identified, including blaOXA-234 and a novel blaOXA-type 350

blaOXA-425 in CRAB and novel blaOXA-type blaOXA-424 and blaOXA-426 in two 351

susceptible strains. In most CRAB strains (25/32, 78%), blaOXA-51-like variants were 352

associated with upstream ISAba1, but no upstream ISAba1 was detected in all three 353

susceptible strains. Furthermore, the blaOXA-23-containing resistance island was 354

identified in all the 32 CRAB strains, and absent in all susceptible strains. It is very 355

likely that blaOXA-234 or blaOXA-425 contributes to carbapenem resistance in A. 356

baumannii, as demonstrated previously with blaOXA-23, blaOXA-24, and blaOXA-58, 357

blaOXA-234 or blaOXA-425 was also associated with upstream ISAba1, which has been 358

shown to promote overexpression of OXA (31). 359

Despite extensive research on the virulence potential of A. baumannii, little is 360

known about its true pathogenic potential or virulence repertoire. A. baumannii 361

thrives in hospital settings largely due to its persistence on abiotic surfaces (32). One 362

mechanism for A. baumannii persistence in hospital settings is the presence of a 363

putative tip adhesion gene, csuE. The csuE gene is involved in pilus and biofilm 364

formation (33), and its presence has been associated with the persistence of A. 365

baumannii on abiotic surfaces such as plastic and glass (33). The resistant strains were 366

found to contain two different types of csuE. Most of the strains contained the 367

shortest-type csuE, which was not observed in 10 resistant strains. The second type of 368

csuE was only detected in two Zhejiang strains isolated in 2004, which comprised 369

ISAba125 and a gene cluster containing 16 proteins, when compared with the shortest 370

type. The T6SS secretion system is widespread among Gram-negative bacteria, and 371

can be used for toxicity against other bacteria and eukaryotic cells. The T6SS system 372

has been implicated in the interaction between bacteria as well as between bacteria 373

and their hosts. In Vibrio cholerae, the T6SS system confers toxicity toward other 374

bacteria, providing a means of interspecies competition to enhance environmental 375

survival (34). Furthermore, Pseudomonas aeruginosa has been found to activate the 376

T6SS system during infection in patients with cystic fibrosis (35), and also use the 377

T6SS-delivered toxins to actively kill competing bacteria (36, 37). In A. baumannii 378

strains, the T6SS secretion system is conserved and plays a role in competition with 379

other bacterial species (38). In the present study, three different types of T6SS region 380

were noted, with one type being predominantly detected. Two of the three susceptible 381

strains did not exhibit this region and only one strain presented a unique type of T6SS 382

region, which was different from those noted in the carbapenem-resistant strains. Two 383

resistant Zhejiang strains collected in 2004 exhibited the third type of T6SS region, 384

which contained more genes, when compared with the predominant type, including 385

VgrG, ATP synthase, and transcriptional regulators. 386

A. baumannii showed a strong ability to acquire foreign DNA such as drug 387

resistance and pathogenicity DNA, which allows the bacterium to acquire genetic 388

diversity and overcome antibiotic selection pressure. The flow of genetic information 389

between the bacterial cells by HGT drives bacterial evolution, and RM systems are 390

the key moderators of this process (39). The presence of DNA RM system may act as 391

a “selfish” genetic element to ensure its dissemination and/or may function in 392

bacteriophage defense and hindrance of lateral gene transfer (40). In the present study, 393

based on the phylogenetic tree, two groups of strains with different founder strains 394

were identified among CRAB. Furthermore, six strains clustered with two strains 395

from Zhejiang isolated in 2004 contained more number of RM system genes. The 396

transfer of resistance genes may facilitate the rapid spread of these genes among A. 397

baumannii strains, so the antimicrobial susceptibility profile of this pathogen should 398

be closely monitored. We speculate that new antibiotics with high ability to interfere 399

the horizontal transfer of the resistance genes or elements carrying them may 400

contribute to effective treatment of Acinetobacter infections in the future. 401

In conclusion, in the present study, we analyzed the genome of 35 representative 402

A. baumannii isolates from China. Extensive variation in the gene content was found, 403

even among strains that were phylogenetically and epidemiologically very closely 404

related. In the process of evolution, the genome of A. baumannii gradually tended to 405

diversify. Several mechanisms contributed to this diversity, including IS-mediated 406

deletions, genome-wide homologous recombination, transfer of mobile genetic 407

elements, and mobilization of transposons or ISs. Thus, the present study improves 408

our understanding of the evolutionary processes that contribute to the emergence of 409

CRAB in China. Future large scale sampling across different areas and time scales are 410

still needed to fully understand the evolution of A. baumannii and its drug resistant 411

development and trends 412

413

ACKNOWLEDGMENTS 414

This study was supported by the National Natural Science Foundation of China (No. 415

31170125), Beijing Natural Science Foundation (5122041), the Specialized Research 416

Fund for the Doctoral Program of Higher Education (20110001110043), and Key 417

Projects in the National Science & Technology Pillar Program (2012EP001002). 418

419

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540

Figure legends: 541

Fig. 1. (A) Whole genome phylogeny of 35 A. baumannii and 17 sequenced A. 542

baumannii genomes. The phylogeny tree was constructed based on SNPs and rooted 543

with A. baylyi ADP1. Colored circles indicate different locations of the strains isolated. 544

Blue triangles indicate that the strains were susceptible to carbapenems. Predominant 545

MLST ST92 is highlighted in red. (B) Results of eBURST analysis conducted to 546

assign CCs in the 35 A. baumannii strains utilizing seven loci. The CCs are circled 547

and the predicted clonal ancestors are shown by the central points. 548

Fig 2. (A) Structure and distribution of the resistance abaR islands. (B) Evolutionary 549

ideograph of the abaR islands in the evolution of CRAB. 550

Fig 3. Transposons containing the drug resistance gene in different A. baumannii 551

strains. (A) Blue and green colors indicate ISAba1, orange color indicates ISAba1 and 552

ISAba16, yellow color indicates ISAba14 and ISAba16, gray color indicates ISAba20 553

and ISAba1, rose-red color indicates ISAba1 and ISAba13, pink color indicates 554

ISAba1 and ISAba18, purple color indicates ISAba1 and ISAba14, and blank indicates 555

the absence of these transposons containing the drug resistance gene. (B) Structures of 556

transposons containing blaOXA23, Tn2009-like (blue in A), and Tn2006 (green in A). 557

Fig 4. Genome features of the 35 A. baumannii strains. (A) Number of total features 558

in the core (green) and pan-(blue) genome as a function of the number of strains 559

sequenced. An average of 500 random permutations of the genome order is presented 560

for the pan- and core genome content; the error bars represent the standard deviation 561

of these results. (B) Intrinsic chromosomal-lactamase alleles. Different colors 562

represent different types; blue color indicates that blaADC is an ADC30 variant and 563

blaOXA66 variant. Horizontal bars indicate the presence of an upstream ISAba1. (C) 564

Variation in the presence of a T6SS gene cluster and csuE gene cluster. Different 565

colors represent different types, and blank indicates that the region is absent. 566

Horizontal bar represents ISAba1 or ISAba13 insertion locations within each locus. T 567

represents transposase insertion locations within each locus. (D) Surface 568

polysaccharide variants for the OC and capsular polysaccharide (K) loci. Different 569

colors represent different types. Horizontal bar represents ISAba1 insertion locations 570

within each locus. (E) Variant region showing recombination around the heme 571

utilization region. Orange color indicates that the heme region is present and blank 572

indicates that the region is absent. (F) RM system. Blue color indicates that the 573

subunit is present and blank indicates that the subunit is absent. (G) Phage content 574

refers to the phage-related region located in TYTH; blue color indicates that both the 575

phages are present, green color indicates that the first phage is present, and other 576

colors indicate that different phage is present (refer to the text for details). 577

578