AAC Accepts, published online ahead of print on 8 December 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.04609-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Evolution of carbapenem-resistant Acinetobacter baumannii through whole
2
genome sequencing and comparative genomic analysis
3 4
Henan Lia#, Fei Liub#, Yawei Zhanga, Xiaojuan Wanga, Chunjiang Zhaoa, Hongbin
5
Chena, Feifei Zhanga, Baoli Zhub, Yongfei Hub*, Hui Wanga*
6 7 8
Department of Clinical Laboratory, Peking University People’s Hospital, Beijing,
9
Chinaa; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
10
Microbiology, Chinese Academy of Sciences, Beijing, Chinab
11 12
Running title: Evolution of carbapenem-resistant A. baumannii
13
Keywords: Acinetobacter baumannii; comparative genomics; antibiotic resistance;
14
evolution.
15 16
#
Henan Li and Fei Liu contributed equally to this work.
17
*
Corresponding authors:
18
Prof. Hui Wang, Department of Clinical Laboratory, Peking University People’s
19
Hospital, Beijing, 100044, China, Tel. +86-10-88326300; Email: [email protected]
20
(H. Wang).
21
Yongfei Hu, Key Laboratory of Pathogenic Microbiology and Immunology, Institute
22
of
23
+86-10-64807433; Email: [email protected] (Y. Hu).
24
Microbiology,
Chinese
Academy
of
Sciences,
Beijing,
China,
Tel.
25
ABSTRACT Acinetobacter baumannii is a globally important nosocomial pathogen
26
characterized by an evolving multidrug resistance. A total of 35 representative clinical
27
A. baumannii strains isolated from 13 hospitals in nine cities of China during
28
1999–2011, including 32 carbapenem-resistant and three susceptible A. baumannii,
29
were selected for whole genome sequencing and comparative genomic analysis.
30
Phylogenetic analysis revealed that the earliest strain 1999BJAB11 and two Zhejiang
31
strains isolated in 2004 were the founder strains of carbapenem-resistant A. baumannii.
32
Ten types of resistance abaR islands were identified, and a previously unreported
33
abaR island was identified in two Zhejiang strains isolated in 2004, which comprised
34
two-component response regulator, resistance-related proteins, and RND efflux
35
system proteins. Multiple transposons or insertion sequences (ISs) existed in each
36
strain, which gradually tended to diversify with evolution. Some of these IS elements
37
or transposons were the first to be reported, and most of them were mainly found in
38
strains from two provinces. Genome feature analysis illustrated diversified resistance
39
genes, surface polysaccharides, and restriction-modification system, even in strains
40
that were phylogenetically and epidemiologically very closely related. IS-mediated
41
deletions
42
lipooligosaccharide (LOS) loci. Recombination occurred in the heme utilization
43
region and intrinsic resistance genes blaADC and blaOXA-51-like variants, and three novel
44
blaOXA-51-like variants, blaOXA-424, blaOXA-425, and blaOXA-426, were identified. Our results
45
could improve the understanding of the evolutionary processes that contribute to the
46
emergence of carbapenem-resistant A. baumannii, and help in elucidating the
47
molecular evolutionary mechanism in A. baumannii.
48 49
were
identified
in
the
T6SS
region,
csuE
region,
and
core
50
Acinetobacter baumannii is an important opportunistic pathogen that has caused
51
severe nosocomial infections worldwide(1). Multidrug-resistant A. baumannii,
52
resistant to carbapenems, has been increasingly reported worldwide, which raises
53
serious concerns about the limited antimicrobial treatment options available (2). Our
54
previous study indicated that the percentage of imipenem- and meropenem-resistant A.
55
baumannii had increased from 4.5% to 61.7% and 4.5% to 62.8% during 2003–2010
56
in China. In 2012, according to the Chinese Meropenem Surveillance Study (CMSS),
57
the susceptibility rates of A. baumannii to imipenem and meropenem were 37.8% and
58
36.0%, respectively (3).
59
A. baumannii rapidly develops multidrug resistance due to the presence of mobile
60
genetic elements, such as insertion sequences (ISs), transposons, and resistance
61
islands (4). Many recent studies have highlighted the diversity in the genomic location,
62
architecture, and content of resistance islands, demonstrating the dynamic nature of A.
63
baumannii antibiotic resistance mechanisms and the adaptive significance of these
64
elements (5-7). However, evolution of antibiotic resistance over time in relation to
65
changes in the major clones in China has not yet been investigated. Moreover, only a
66
few studies have focused on the genetic background differences among A. baumannii
67
isolates collected from different locations at different time periods.
68
Comparative genomics study helps in the evaluation of the resistance
69
mechanisms, pathogenicity, and evolution of bacterial pathogens at genome-wide
70
level (8). Despite increased research on A. baumannii epidemiology and evolution
71
(9-12), large gaps remain in our understanding of the evolutionary processes that
72
contribute to multidrug resistance and genomic diversification of A. baumannii,
73
especially in China. In the present study, a total of 35 representative A. baumannii
74
strains isolated from 13 hospitals in 9 cities of China during 1999–2011 were selected
75
for comparative whole genome sequencing to examine strain-level genetic diversity
76
and gene content variation. Through the analysis of these strains with different
77
backgrounds, we aimed to gain a better understanding of the resistance evolution of A.
78
baumannii in China.
79
MATERIALS AND METHODS
80
Strain isolation and genotypic and phenotypic characterization. The A. baumannii
81
strains were collected from 13 hospitals in nine cities of China from 1999 to 2011.
82
The isolates from 1999 to 2005 were selected from 221 carbapenem-resistant A.
83
baumannii (CRAB) in our previous study (13), and the isolates from 2011 were
84
selected from 283 A. baumannii strains in the Chinese Antimicrobial Resistance
85
Surveillance of Nosocomial infections (CARES) (14). The isolates were subjected to
86
additional analysis to characterize the antibiotic phenotypes (Table S1) and genotypes
87
via MLST (http://pubmlst.org/abaumannii/) (15). From this collection of isolates, 35
88
representative isolates were selected from predominant clone in different locations,
89
dates, and sources. To put the sequenced strains in a phylogenetic context and assess
90
the shared and clade-specific gene contents, the genomes were compared with 17
91
reference Acinetobacter genomes available in NCBI as of January 2014.
92
DNA preparation, library construction, sequencing, and assembly. DNA was
93
isolated with the DNA purification kit (Qiagen). Illumina sequencing libraries were
94
prepared by using Nextera kits with indexed-encoded adapters from Illumina,
95
according to the manufacturer’s instructions. The libraries were pooled for sequencing
96
on MiSeq, and paired-end sequence reads were obtained representing 50–200-fold
97
genome coverage. The Illumina sequence data were assembled using SOAP denovo
98
(Version 2.04). PCR amplification and Sanger sequencing were used to solve the
99
ambiguity of the order and orientation of scaffolds.
100
Genome annotation. The assembled genome sequence was annotated by the
101
NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), using Glimmer
102
3.0 for the identification of protein-coding genes (16), tRNAscan-SE for tRNA genes
103
(17), and RNAmmer for rRNA genes (18). The ISs were identified using the IS Finder
104
database (www-is.biotoul.fr) (19). The origin of replication (oriC) and putative DnaA
105
boxes were identified using Ori-Finder (20).
106
Whole genome phylogeny construction. Multiple sequence alignments of 52
107
genomes were performed using Mugsy (21).The tree was constructed based on SNPs
108
from the whole genome alignment. This alignment included SNPs from 17 reference
109
genomes and 35 genomes sequenced in this study.
110
eBURST analysis. eBURST analysis was employed to investigate the
111
evolutionary relationships and CCs within the isolates, using the software on the
112
eBURST website (http://eburst.mlst.net/v3/enter_data/single/), with statistical support
113
for the complexes being assessed via the bootstrap resampling method with 1000
114
resamplings (22). The analysis was performed using both stringent (a minimum of six
115
shared alleles) and relaxed (a minimum of five shared alleles) grouping parameters.
116
Estimation of core and pan-genome size. A BLASTP search between every pair
117
of protein sequences from each strain was performed. PanOCT was used to identify
118
the orthologs with the BLASTP output (23).
119
Nucleotide sequence accession numbers. This Whole Genome Shotgun project
120
has been deposited at DDBJ/EMBL/GenBank under the accession PRJNA261018.
121
The three novel blaOXA types were designated as blaOXA-424 (GenBank accession
122
number: KM588352), blaOXA-425 (GenBank accession number: KM588353), and
123
blaOXA-426 (GenBank accession number: KM588354) (http://www.lahey.org/Studies/).
124
125
RESULTS
126
A total of 35 representative A. baumannii, including 32 carbapenem-resistant A.
127
baumannii (CRAB) and three susceptible A. baumannii, isolated from patients with
128
hospital-acquired infections in 13 hospitals in nine cities of China during 1999–2011
129
were selected for whole genome sequencing. The 32 CRAB isolates consisted of one
130
isolate from 1999, eight isolates from 2003–2005, and 23 isolates from 2011. Most of
131
the isolates were collected in Beijing, China (14/35, 40%). The sources of these 35
132
isolates were sputum (20/35, 57.1%), blood (10/35, 28.6%), and abdominal fluid
133
(5/35, 14.3%). The general characteristics of the 35 A. baumannii genomes are listed
134
in Table S2.
135
Whole genome phylogeny of the genus Acinetobacter. To facilitate detailed
136
analysis of strain relationships, we developed a phylogeny based on single nucleotide
137
polymorphisms (SNPs) from whole genome alignment to represent the ancestral
138
relationships among 35 strains and 16 other A. baumannii strains (Fig. 1A). These
139
included 12 multidrug-resistant A. baumannii strains (AYE, AB0057, ACICU,
140
AB1656-2, TYTH-1, TCDC-AB0715, BJAB07104, BJAB0715, BJAB0868, ZW85-1,
141
MDR-TJ, and MDR-ZJ06), two susceptible strains (ATCC17978 and AB307-0294),
142
one community-acquired strain D1279779, and one non-clinical strain SDF isolated
143
from a human body louse. ADP1, a soil-living Acinetobacter baylyi strain was used as
144
the outgroup for comparison.
145
Based on the phylogenetic data, all the strains, along with nine previously
146
reported Asian strains, including MDR-ZJ06, MDR-TJ, ZW85-1, BJAB07104,
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BJAB0715, and BJAB0868 from mainland China, TCDC-AB0715 and TYTH-1 from
148
China, Taiwan, and AB1656-2 from Korea, were grouped together with ACICU, a
149
strain of global clone II (GC II) group. The susceptible strains were clustered on the
150
edge of the evolutionary tree. Two groups of founder strains were identified among
151
the CRAB based on the phylogenetic tree, which were the earliest isolated strain
152
1999BJAB11 and two strains from Zhejiang isolated in 2004. Interestingly, the
153
earliest isolated strain 1999BJAB11 was separated from all of the other ST92 strains,
154
suggesting that it may have a different genome content, when compared with the other
155
ST92 strains.
156
The eBURST algorithm revealed one clonal complex (CC) CC1 and six
157
singletons (Fig. 1B). The CC1 contained ST92, which was identified as a potential
158
founder, with ST75, ST137, ST90, ST118, and ST138 radiating from it. All the
159
isolates in CC1 were CRAB, and the three susceptible strains were clustered into
160
singletons. The location and source of these isolates showed diversity in different ST.
161
Evolution of the resistance abaR islands. Ten types of the resistance abaR
162
islands were identified in the 32 CRAB strains (Fig. 2A). Except for the type 6 abaR
163
island, other types shared high homology. Type 10 abaR island was the predominant
164
type, which was identified in 12 strains (12/32, 37.5%), and was 19.123 kb in length
165
and contained the highest number of resistance genes, including tniB, usp, sul, tetA,
166
tetR, arsR, and aph, similar to those noted in a strain from Japan with one different
167
nucleotide (24). Type 5 abaR island in 1999BJAB11 contained the largest number
168
genes, and the conjugal transfer gene traA was identified only in this isolate. The
169
resistance genes tetA, tetR, arsR, aphA, and strB were not found in type 1 abaR island.
170
While
171
phosphoglucosamine mutase (glmM) were detected in type 2, 7, and 10 abaR islands.
172
The phosphoglucosamine mutase (glmM) can catalyze the conversion of
173
glucosamine-6-phosphate to glucosamine-1-phosphate, which is an essential step in
174
the formation of the cell wall precursor UDP-N-acetylglucosamine (25). In
175
Streptococcus gordonii, mutations in glmM appear to influence bacterial cell growth
176
and morphology, biofilm formation, and sensitivity to penicillins (26). Function of
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glmM in A. baumannii was currently not well known. The mobile element protein was
178
not observed in type 3 and 10 abaR islands. Two strains from Zhejiang isolated in
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2011 contained type 9 abaR island, which showed gene arrangement when compared
180
with type 8 abaR island. Two Zhejiang strains collected in 2004 had the type 6 abaR
181
island, which included the two-component response regulator, osmosensitive K+
182
channel histidine kinase KdpD, methyl viologen resistance protein smvA, RND
183
multidrug efflux transporter acriflavin resistance protein, and RND efflux system
184
membrane fusion protein CmeA. These genes were not observed in the other nine
the
sulfonamide
resistant
protein
dihydropteroate
synthase
and
185
types of abaR islands. The evolutionary ideograph of the abaR islands is shown in Fig.
186
2B, which was according to the time. The type 5 abaR island in the earliest strain
187
1999BJAB11 contained the largest number genes, and with the genome evolution,
188
gene acquirement, gene loss and gene rearrangement were detected in the strains
189
isolated during 2003-2011. The type 6 abaR island in two Zhejiang strains was
190
relatively independent of the other strains during abaR islands evolution.
191
Variations in transposon-associated antibiotic resistance genes. The
192
transposons containing drug resistance genes varied in different A. baumannii strains
193
(Fig. 3A). BlaOXA-51-like gene was located in three types of transposons. Eleven strains
194
possessed IS-blaOXA-51-like-IS transposon, and 10 of them were flanked by two ISAba1
195
elements. One strain 2011ZJAB2 was flanked by ISAba1 and ISAba16, and eight
196
strains possessed ISAba1-blaOXA-51-like-metallo-beta-lactamase-ISAba1 transposons.
197
Only
198
class-A-beta-lactamase-ISAba1 transposon. ISAba1-metallo-beta-lactamase-ISAba1
199
was detected in 11 stains, but in two strains from Zhejiang isolated in 2011,
200
metallo-beta-lactamase
201
ISAba1-class-A-beta-lactamase-ISAba1 was identified in 13 strains. In one strain
202
from Zhejiang isolated in 2004, ISAba20 and ISAba1 were detected on both the sides
203
of class A beta-lactamase; and in two strains from Guangdong isolated in 2011,
204
ISAba1 and ISAba13 were detected. In seven strains isolated in 2011, blaADC-30 was
205
located in ISAba1-blaADC-30-ISAba1, but in 2005LNAB4, blaADC-30 was flanked by
206
ISAba1 and ISAba18, and in two strains from Zhejiang isolated in 2011, blaADC-30 was
207
flanked by ISAba1 and ISAba14. Furthermore, in two strains from Zhejiang isolated
208
in 2004, ISAba1-ampC-ISAba1 was identified.
one
strain
2011LNAB2
was
flanked
possessed
by
ISAba14
ISAba1-blaOXA-51-like-
and
ISAba16.
209
The blaOXA-23-containing resistance island was identified in all the 32 CRAB
210
strains (Fig. 3B). In most of the sequenced strains (30/32, 93.8%), blaOXA-23 was
211
located in a transposon, which showed 99% identity to Tn2009. Tn2009 was initially
212
identified on the chromosome of A. baumannii MDR-ZJ06 from China, with a length
213
of 8421 bp and flanked by two ISAba1 elements. Tn2009 was noted to carry the
214
blaOXA-23 gene, the truncated DEAD/DEAH box helicase gene, the ATPase gene, the
215
yeeC gene, and the yeeB gene. However, in two isolates from Guangzhou obtained in
216
2011, blaOXA-23 was found in transposon Tn2006, which was shorter than Tn2009.
217
Tn2006 contained the ATPase gene, the yeeA gene, and the yeeB gene, and was
218
flanked by two ISAba1 elements.
219
Pan-genome: Core and accessory genes. All the sequenced A. baumannii
220
strains contained a genetically highly homogeneous core genome that encodes
221
proteins involved in DNA replication, transcription, and translation as well as many
222
metabolic pathways. By using the best reciprocal BLAST matches, we identified 2830
223
core genes/proteins and 1206 accessory genes/proteins among all the 35 A. baumannii
224
isolates. Graphing of the numbers of core and pan-genome as a function of the
225
number of strains sequenced revealed that the slope for the core gene number was
226
approaching an asymptote, whereas the pan-genome continued to expand even after
227
compilation of 35 genomes (Fig. 4A).
228
Variation in intrinsic chromosomal resistance genes related to carbapenem
229
resistance. The phylogenetic distribution of different alleles of blaOXA-51-like and
230
blaADC showed evidence of recombination and mutation. The presence of an upstream
231
insertion element, ISAba1, suggested that the entire region was replaced by
232
homologous recombination. The blaADC gene was always associated with upstream
233
ISAba1 oriented to allow overexpression of the gene, except in three susceptible
234
strains (Fig. 4B). The sequences from 19 strains were identical to the
235
extended-spectrum blaADC-30 variant (blue), and three strains showed 99% identity to
236
blaADC-30 (dark blue). Five strains contained blaADC-25 (yellow), and one strain carried
237
blaADC-56 (purple). One susceptible strain 2011BJAB9 and three resistant strains
238
showed 99% identity to blaADC-53 (orange), while blaADC in 1999BJAB1 showed 99%
239
identity to blaADC-29. The sequences from the susceptible strains 2011GDAB2 and
240
2011TJAB1 showed 98% and 99% identity to blaADC-52 and blaADC-63, respectively.
241
The blaOXA-51-like variants were different in the resistant strains and susceptible
242
strains. The most common blaOXA-51-like variant in resistant strains was blaOXA-66,
243
which was detected in 27 resistant strains (27/35, 77.1%), and blaOXA-68 were detected
244
in three resistant strains from Zhejiang. One unusual variant blaOXA-234 was detected
245
in a resistant strain from Jiangsu in 2005. Furthermore, three novel variants were
246
identified, including blaOXA-425 in the resistant strain 2011BJAB7 and blaOXA-424 and
247
blaOXA-426 in the susceptible strains 2011GDAB2 and 2011BJAB9, respectively. In the
248
susceptible strain 2011TJAB1, blaOXA-69 was detected. In most CRAB strains (25/32,
249
78%), blaOXA-51-like variants were associated with upstream ISAba1, but no upstream
250
ISAba1 was detected in all three susceptible strains.
251
IS-mediated T6SS and csuE region deletions. There were multiple instances of
252
chromosomal gene loss that might have possibly been mediated by an IS adjacent to
253
the deletion (Fig. 4C). Two large deletions adjacent to ISAba1 elements have been
254
previously described (10), including a 40-kbp region encoding the entire type VI
255
secretion system (T6SS) and a ~20-kbp region of adhesion genes (csuE) involved in
256
aspartate metabolism. In the present study, three different types of T6SS region were
257
noted, and eight strains lacked this region. Among these eight strains, in five strains,
258
deletion was mediated by IS insertion. It is worth noting that except for ISAba1
259
insertion in three strains isolated in 2011, ISAba13 insertion was detected in two
260
strains from Guangdong isolated in 2011. Similarly, three different types of csuE
261
region were also noted, and 12 strains lacked this region. Among these 12 strains,
262
deletion was mediated by IS insertion in three strains and transposase insertion in two
263
strains, respectively. Except for ISAba1-mediated deletion in 2011BJAB7, this study
264
is the first to identify ISAba125 insertion in two strains from Zhejiang isolated in
265
2004 and transposase insertion in the csuE region in two strains from Guangdong
266
isolated in 2011.
267
Variation in surface polysaccharide synthesis and heme utilization region.
268
The 35 strains showed substantial variation in the content and organization of loci
269
involved in surface polysaccharide synthesis (Fig. 4D). The core lipooligosaccharide
270
(LOS) loci (outer core [OC] locus) (27) consisted of one predominant type (blue),
271
which was present in most of the strains (29/35, 82.9%); however, we also identified
272
several variations in the three susceptible strains and one resistant strain. In two
273
strains from Zhejiang isolated in 2004, ISAba20 insertions were detected. With regard
274
to capsular (K locus) loci, 12 variations were identified. One predominant type (blue)
275
existed in 10 strains (28.6%), which was most closelgy related to ACICU (99.5%
276
identity at the nucleotide level).
277
The heme utilization region, containing several genes involved in heme
278
utilization, was more common in strains collected in 2011. However, this region was
279
absent in 1999BJAB1 and most of the strains collected during 2003–2005, except for
280
two strains from Zhejiang isolated in 2004. Among the 23 strains collected in 2011,
281
the heme utilization region was noted in 18 strains, which was 96% identical to
282
ACICU (Fig. 4E).
283
Variation in the restriction-modification system. The restriction-modification
284
(RM) system is a major participant in the co-evolutionary interaction between mobile
285
genetic elements (MGEs) and their hosts through the regulation of horizontal gene
286
transfer (HGT). In the present study, six strains clustered with two strains from
287
Zhejiang isolated in 2004 contained more RM system genes (Fig. 4F). In the
288
susceptible strain 2011TJAB1 and two resistant strains from Zhejiang isolated in 2004,
289
we identified Type I RM system methylase subunit, restriction subunit, and specificity
290
subunit. Furthermore, strains 2011ZJAB1 and 2011BJAB3 only had Type I RM
291
system methylase subunit, and strain 2011ZJAB4 only had Type I RM system
292
restriction subunit. Type II RM system methylase subunit was detected in strain
293
1999BJAB11 and susceptible strain 2011TJAB1, whereas Type III RM system
294
methylation subunit was found in three Zhejiang strains and two Guangdong strains.
295
CRISPR-associated protein was only detected in one susceptible strain 2011BJAB9.
296
Variation in phage-related regions. Phage-related regions are another source of
297
variability contributing to the difference among A. baumannii strains. Two
298
predominant types of phage-related regions were located within the same
299
chromosomal location corresponding to 1.11–1.16 Mbp in ACICU (ACICU_00997 to
300
ACICU_01077) and 1.45–1.53 Mbp in TYTH-1 (M3Q_1334 to M3Q_1458) (Fig.
301
4G). One primary variant typified by the completed reference genome of TYTH-1
302
consisted of two probable phage insertion events flanked by phage integrases (blue),
303
whereas another variant possessed only the second of the two phage elements (green).
304
Two susceptible strains 2011TJAB1 and 2011BJAB9 and strain 2011ZJAB4 had
305
different phage-related regions, when compared with the two main variants, and all
306
these strains were susceptible to tigecycline.
307
308
DISCUSSION
309
Despite increased research on A. baumannii evolution, the development of multidrug
310
resistance and genomic diversification of A. baumannii is not yet clear, especially in
311
China. In the present study, we have reported the whole genome sequences and
312
comparative genomic analysis of 35 representative A. baumannii strains isolated
313
during 1999–2011 in 13 hospitals in nine cities in China, including 32 CRAB and
314
three susceptible A. baumannii with different genotypes and phenotypes. These
315
genomes represent a significant addition of genomic data from the genus and could
316
serve as a valuable resource for future genomic studies.
317
It has been indicated that the resistance abaR islands have evolved into diverse
318
types through multiple events of insertion, deletion, and recombination (6, 28). The
319
predominance of type 4 abaR resistance islands among the CRAB isolates have been
320
reported in South Korea (29) and Taiwan (30). However, the evolution of resistance
321
abaR islands has not yet been investigated, especially in China. In the present study, it
322
was found that the type 6 abaR island in two Zhejiang strains collected in 2004 is
323
different from the previously identified abaR islands, which may thus be a novel abaR
324
island. The type 6 abaR island was noted to comprise the two-component response
325
regulator, osmosensitive K+ channel histidine kinase KdpD, methyl viologen
326
resistance protein smvA, RND multidrug efflux transporter acriflavin resistance
327
protein, and RND efflux system membrane fusion protein CmeA. These regulators
328
and resistance-related proteins in the type 6 abaR island may enhance the strain’s
329
adaptability and antibiotic resistance, which should be further studied.
330
Frequently encountered transposons and ISs carrying resistance genes in bacteria
331
reflect recombination flexibility during acquisition of resistance (4). In the present
332
study, each strain contained multiple transposons or ISs, which gradually tended to
333
diversify in the process of evolution. It must be noted that some of these ISs or
334
transposons are novel, and that most of the novel IS element insertion or transposons
335
were mainly found in strains from Zhejiang or Guangdong province of China. The
336
resistance genes or elements may spread through a transposon- or IS-mediated
337
mechanism, and the diversification of transposons or ISs in the process of evolution
338
increases the possibility of acquisition of novel resistance genes or elements.
339
Recombination has been reported to contribute to the change in the A. baumannii
340
genome (28). Furthermore, the heme utilization region is also highly variable among
341
the A. baumannii strains, which was more common in strains collected in 2011 in the
342
present study, thus making it difficult to assess whether this region was present in a
343
common ancestor and lost by some strains or was gained and subsequently transferred
344
via recombination to different lineages as initially observed. In addition, evidence of
345
recombination events in the allelic distribution of blaADC and blaOXA-51-like variants
346
was also noted. In all CRAB strains, the blaADC gene was associated with upstream
347
ISAba1 oriented to allow overexpression of the blaADC genes. However, in all three
348
susceptible strains, no upstream ISAba1 was detected. In addition, the blaOXA-51-like
349
variants were different in the resistant strains and susceptible strains. Four uncommon
350
blaOXA-51-like genes were identified, including blaOXA-234 and a novel blaOXA-type
351
blaOXA-425 in CRAB and novel blaOXA-type blaOXA-424 and blaOXA-426 in two
352
susceptible strains. In most CRAB strains (25/32, 78%), blaOXA-51-like variants were
353
associated with upstream ISAba1, but no upstream ISAba1 was detected in all three
354
susceptible strains. Furthermore, the blaOXA-23-containing resistance island was
355
identified in all the 32 CRAB strains, and absent in all susceptible strains. It is very
356
likely that blaOXA-234 or blaOXA-425 contributes to carbapenem resistance in A.
357
baumannii, as demonstrated previously with blaOXA-23, blaOXA-24, and blaOXA-58,
358
blaOXA-234 or blaOXA-425 was also associated with upstream ISAba1, which has been
359
shown to promote overexpression of OXA (31).
360
Despite extensive research on the virulence potential of A. baumannii, little is
361
known about its true pathogenic potential or virulence repertoire. A. baumannii
362
thrives in hospital settings largely due to its persistence on abiotic surfaces (32). One
363
mechanism for A. baumannii persistence in hospital settings is the presence of a
364
putative tip adhesion gene, csuE. The csuE gene is involved in pilus and biofilm
365
formation (33), and its presence has been associated with the persistence of A.
366
baumannii on abiotic surfaces such as plastic and glass (33). The resistant strains were
367
found to contain two different types of csuE. Most of the strains contained the
368
shortest-type csuE, which was not observed in 10 resistant strains. The second type of
369
csuE was only detected in two Zhejiang strains isolated in 2004, which comprised
370
ISAba125 and a gene cluster containing 16 proteins, when compared with the shortest
371
type. The T6SS secretion system is widespread among Gram-negative bacteria, and
372
can be used for toxicity against other bacteria and eukaryotic cells. The T6SS system
373
has been implicated in the interaction between bacteria as well as between bacteria
374
and their hosts. In Vibrio cholerae, the T6SS system confers toxicity toward other
375
bacteria, providing a means of interspecies competition to enhance environmental
376
survival (34). Furthermore, Pseudomonas aeruginosa has been found to activate the
377
T6SS system during infection in patients with cystic fibrosis (35), and also use the
378
T6SS-delivered toxins to actively kill competing bacteria (36, 37). In A. baumannii
379
strains, the T6SS secretion system is conserved and plays a role in competition with
380
other bacterial species (38). In the present study, three different types of T6SS region
381
were noted, with one type being predominantly detected. Two of the three susceptible
382
strains did not exhibit this region and only one strain presented a unique type of T6SS
383
region, which was different from those noted in the carbapenem-resistant strains. Two
384
resistant Zhejiang strains collected in 2004 exhibited the third type of T6SS region,
385
which contained more genes, when compared with the predominant type, including
386
VgrG, ATP synthase, and transcriptional regulators.
387
A. baumannii showed a strong ability to acquire foreign DNA such as drug
388
resistance and pathogenicity DNA, which allows the bacterium to acquire genetic
389
diversity and overcome antibiotic selection pressure. The flow of genetic information
390
between the bacterial cells by HGT drives bacterial evolution, and RM systems are
391
the key moderators of this process (39). The presence of DNA RM system may act as
392
a “selfish” genetic element to ensure its dissemination and/or may function in
393
bacteriophage defense and hindrance of lateral gene transfer (40). In the present study,
394
based on the phylogenetic tree, two groups of strains with different founder strains
395
were identified among CRAB. Furthermore, six strains clustered with two strains
396
from Zhejiang isolated in 2004 contained more number of RM system genes. The
397
transfer of resistance genes may facilitate the rapid spread of these genes among A.
398
baumannii strains, so the antimicrobial susceptibility profile of this pathogen should
399
be closely monitored. We speculate that new antibiotics with high ability to interfere
400
the horizontal transfer of the resistance genes or elements carrying them may
401
contribute to effective treatment of Acinetobacter infections in the future.
402
In conclusion, in the present study, we analyzed the genome of 35 representative
403
A. baumannii isolates from China. Extensive variation in the gene content was found,
404
even among strains that were phylogenetically and epidemiologically very closely
405
related. In the process of evolution, the genome of A. baumannii gradually tended to
406
diversify. Several mechanisms contributed to this diversity, including IS-mediated
407
deletions, genome-wide homologous recombination, transfer of mobile genetic
408
elements, and mobilization of transposons or ISs. Thus, the present study improves
409
our understanding of the evolutionary processes that contribute to the emergence of
410
CRAB in China. Future large scale sampling across different areas and time scales are
411
still needed to fully understand the evolution of A. baumannii and its drug resistant
412
development and trends
413
414
ACKNOWLEDGMENTS
415
This study was supported by the National Natural Science Foundation of China (No.
416
31170125), Beijing Natural Science Foundation (5122041), the Specialized Research
417
Fund for the Doctoral Program of Higher Education (20110001110043), and Key
418
Projects in the National Science & Technology Pillar Program (2012EP001002).
419
420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463
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541
Figure legends:
542
Fig. 1. (A) Whole genome phylogeny of 35 A. baumannii and 17 sequenced A.
543
baumannii genomes. The phylogeny tree was constructed based on SNPs and rooted
544
with A. baylyi ADP1. Colored circles indicate different locations of the strains isolated.
545
Blue triangles indicate that the strains were susceptible to carbapenems. Predominant
546
MLST ST92 is highlighted in red. (B) Results of eBURST analysis conducted to
547
assign CCs in the 35 A. baumannii strains utilizing seven loci. The CCs are circled
548
and the predicted clonal ancestors are shown by the central points.
549
Fig 2. (A) Structure and distribution of the resistance abaR islands. (B) Evolutionary
550
ideograph of the abaR islands in the evolution of CRAB.
551
Fig 3. Transposons containing the drug resistance gene in different A. baumannii
552
strains. (A) Blue and green colors indicate ISAba1, orange color indicates ISAba1 and
553
ISAba16, yellow color indicates ISAba14 and ISAba16, gray color indicates ISAba20
554
and ISAba1, rose-red color indicates ISAba1 and ISAba13, pink color indicates
555
ISAba1 and ISAba18, purple color indicates ISAba1 and ISAba14, and blank indicates
556
the absence of these transposons containing the drug resistance gene. (B) Structures of
557
transposons containing blaOXA23, Tn2009-like (blue in A), and Tn2006 (green in A).
558
Fig 4. Genome features of the 35 A. baumannii strains. (A) Number of total features
559
in the core (green) and pan-(blue) genome as a function of the number of strains
560
sequenced. An average of 500 random permutations of the genome order is presented
561
for the pan- and core genome content; the error bars represent the standard deviation
562
of these results. (B) Intrinsic chromosomal-lactamase alleles. Different colors
563
represent different types; blue color indicates that blaADC is an ADC30 variant and
564
blaOXA66 variant. Horizontal bars indicate the presence of an upstream ISAba1. (C)
565
Variation in the presence of a T6SS gene cluster and csuE gene cluster. Different
566
colors represent different types, and blank indicates that the region is absent.
567
Horizontal bar represents ISAba1 or ISAba13 insertion locations within each locus. T
568
represents transposase insertion locations within each locus. (D) Surface
569
polysaccharide variants for the OC and capsular polysaccharide (K) loci. Different
570
colors represent different types. Horizontal bar represents ISAba1 insertion locations
571
within each locus. (E) Variant region showing recombination around the heme
572
utilization region. Orange color indicates that the heme region is present and blank
573
indicates that the region is absent. (F) RM system. Blue color indicates that the
574
subunit is present and blank indicates that the subunit is absent. (G) Phage content
575
refers to the phage-related region located in TYTH; blue color indicates that both the
576
phages are present, green color indicates that the first phage is present, and other
577
colors indicate that different phage is present (refer to the text for details).
578
1
Evolution of carbapenem-resistant Acinetobacter baumannii through whole
2
genome sequencing and comparative genomic analysis
3 4
Henan Lia#, Fei Liub#, Yawei Zhanga, Xiaojuan Wanga, Chunjiang Zhaoa, Hongbin
5
Chena, Feifei Zhanga, Baoli Zhub, Yongfei Hub*, Hui Wanga*
6 7 8
Department of Clinical Laboratory, Peking University People’s Hospital, Beijing,
9
Chinaa; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
10
Microbiology, Chinese Academy of Sciences, Beijing, Chinab
11 12
Running title: Evolution of carbapenem-resistant A. baumannii
13
Keywords: Acinetobacter baumannii; comparative genomics; antibiotic resistance;
14
evolution.
15 16
#
Henan Li and Fei Liu contributed equally to this work.
17
*
Corresponding authors:
18
Prof. Hui Wang, Department of Clinical Laboratory, Peking University People’s
19
Hospital, Beijing, 100044, China, Tel. +86-10-88326300; Email: [email protected]
20
(H. Wang).
21
Yongfei Hu, Key Laboratory of Pathogenic Microbiology and Immunology, Institute
22
of
23
+86-10-64807433; Email: [email protected] (Y. Hu).
24
Microbiology,
Chinese
Academy
of
Sciences,
Beijing,
China,
Tel.
25
ABSTRACT Acinetobacter baumannii is a globally important nosocomial pathogen
26
characterized by an evolving multidrug resistance. A total of 35 representative clinical
27
A. baumannii strains isolated from 13 hospitals in nine cities of China during
28
1999–2011, including 32 carbapenem-resistant and three susceptible A. baumannii,
29
were selected for whole genome sequencing and comparative genomic analysis.
30
Phylogenetic analysis revealed that the earliest strain 1999BJAB11 and two Zhejiang
31
strains isolated in 2004 were the founder strains of carbapenem-resistant A. baumannii.
32
Ten types of resistance abaR islands were identified, and a previously unreported
33
abaR island was identified in two Zhejiang strains isolated in 2004, which comprised
34
two-component response regulator, resistance-related proteins, and RND efflux
35
system proteins. Multiple transposons or insertion sequences (ISs) existed in each
36
strain, which gradually tended to diversify with evolution. Some of these IS elements
37
or transposons were the first to be reported, and most of them were mainly found in
38
strains from two provinces. Genome feature analysis illustrated diversified resistance
39
genes, surface polysaccharides, and restriction-modification system, even in strains
40
that were phylogenetically and epidemiologically very closely related. IS-mediated
41
deletions
42
lipooligosaccharide (LOS) loci. Recombination occurred in the heme utilization
43
region and intrinsic resistance genes blaADC and blaOXA-51-like variants, and three novel
44
blaOXA-51-like variants, blaOXA-424, blaOXA-425, and blaOXA-426, were identified. Our results
45
could improve the understanding of the evolutionary processes that contribute to the
46
emergence of carbapenem-resistant A. baumannii, and help in elucidating the
47
molecular evolutionary mechanism in A. baumannii.
48 49
were
identified
in
the
T6SS
region,
csuE
region,
and
core
50
Acinetobacter baumannii is an important opportunistic pathogen that has caused
51
severe nosocomial infections worldwide(1). Multidrug-resistant A. baumannii,
52
resistant to carbapenems, has been increasingly reported worldwide, which raises
53
serious concerns about the limited antimicrobial treatment options available (2). Our
54
previous study indicated that the percentage of imipenem- and meropenem-resistant A.
55
baumannii had increased from 4.5% to 61.7% and 4.5% to 62.8% during 2003–2010
56
in China. In 2012, according to the Chinese Meropenem Surveillance Study (CMSS),
57
the susceptibility rates of A. baumannii to imipenem and meropenem were 37.8% and
58
36.0%, respectively (3).
59
A. baumannii rapidly develops multidrug resistance due to the presence of mobile
60
genetic elements, such as insertion sequences (ISs), transposons, and resistance
61
islands (4). Many recent studies have highlighted the diversity in the genomic location,
62
architecture, and content of resistance islands, demonstrating the dynamic nature of A.
63
baumannii antibiotic resistance mechanisms and the adaptive significance of these
64
elements (5-7). However, evolution of antibiotic resistance over time in relation to
65
changes in the major clones in China has not yet been investigated. Moreover, only a
66
few studies have focused on the genetic background differences among A. baumannii
67
isolates collected from different locations at different time periods.
68
Comparative genomics study helps in the evaluation of the resistance
69
mechanisms, pathogenicity, and evolution of bacterial pathogens at genome-wide
70
level (8). Despite increased research on A. baumannii epidemiology and evolution
71
(9-12), large gaps remain in our understanding of the evolutionary processes that
72
contribute to multidrug resistance and genomic diversification of A. baumannii,
73
especially in China. In the present study, a total of 35 representative A. baumannii
74
strains isolated from 13 hospitals in 9 cities of China during 1999–2011 were selected
75
for comparative whole genome sequencing to examine strain-level genetic diversity
76
and gene content variation. Through the analysis of these strains with different
77
backgrounds, we aimed to gain a better understanding of the resistance evolution of A.
78
baumannii in China.
79
MATERIALS AND METHODS
80
Strain isolation and genotypic and phenotypic characterization. The A. baumannii
81
strains were collected from 13 hospitals in nine cities of China from 1999 to 2011.
82
The isolates from 1999 to 2005 were selected from 221 carbapenem-resistant A.
83
baumannii (CRAB) in our previous study (13), and the isolates from 2011 were
84
selected from 283 A. baumannii strains in the Chinese Antimicrobial Resistance
85
Surveillance of Nosocomial infections (CARES) (14). The isolates were subjected to
86
additional analysis to characterize the antibiotic phenotypes (Table S1) and genotypes
87
via MLST (http://pubmlst.org/abaumannii/) (15). From this collection of isolates, 35
88
representative isolates were selected from predominant clone in different locations,
89
dates, and sources. To put the sequenced strains in a phylogenetic context and assess
90
the shared and clade-specific gene contents, the genomes were compared with 17
91
reference Acinetobacter genomes available in NCBI as of January 2014.
92
DNA preparation, library construction, sequencing, and assembly. DNA was
93
isolated with the DNA purification kit (Qiagen). Illumina sequencing libraries were
94
prepared by using Nextera kits with indexed-encoded adapters from Illumina,
95
according to the manufacturer’s instructions. The libraries were pooled for sequencing
96
on MiSeq, and paired-end sequence reads were obtained representing 50–200-fold
97
genome coverage. The Illumina sequence data were assembled using SOAP denovo
98
(Version 2.04). PCR amplification and Sanger sequencing were used to solve the
99
ambiguity of the order and orientation of scaffolds.
100
Genome annotation. The assembled genome sequence was annotated by the
101
NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), using Glimmer
102
3.0 for the identification of protein-coding genes (16), tRNAscan-SE for tRNA genes
103
(17), and RNAmmer for rRNA genes (18). The ISs were identified using the IS Finder
104
database (www-is.biotoul.fr) (19). The origin of replication (oriC) and putative DnaA
105
boxes were identified using Ori-Finder (20).
106
Whole genome phylogeny construction. Multiple sequence alignments of 52
107
genomes were performed using Mugsy (21).The tree was constructed based on SNPs
108
from the whole genome alignment. This alignment included SNPs from 17 reference
109
genomes and 35 genomes sequenced in this study.
110
eBURST analysis. eBURST analysis was employed to investigate the
111
evolutionary relationships and CCs within the isolates, using the software on the
112
eBURST website (http://eburst.mlst.net/v3/enter_data/single/), with statistical support
113
for the complexes being assessed via the bootstrap resampling method with 1000
114
resamplings (22). The analysis was performed using both stringent (a minimum of six
115
shared alleles) and relaxed (a minimum of five shared alleles) grouping parameters.
116
Estimation of core and pan-genome size. A BLASTP search between every pair
117
of protein sequences from each strain was performed. PanOCT was used to identify
118
the orthologs with the BLASTP output (23).
119
Nucleotide sequence accession numbers. This Whole Genome Shotgun project
120
has been deposited at DDBJ/EMBL/GenBank under the accession PRJNA261018.
121
The three novel blaOXA types were designated as blaOXA-424 (GenBank accession
122
number: KM588352), blaOXA-425 (GenBank accession number: KM588353), and
123
blaOXA-426 (GenBank accession number: KM588354) (http://www.lahey.org/Studies/).
124
125
RESULTS
126
A total of 35 representative A. baumannii, including 32 carbapenem-resistant A.
127
baumannii (CRAB) and three susceptible A. baumannii, isolated from patients with
128
hospital-acquired infections in 13 hospitals in nine cities of China during 1999–2011
129
were selected for whole genome sequencing. The 32 CRAB isolates consisted of one
130
isolate from 1999, eight isolates from 2003–2005, and 23 isolates from 2011. Most of
131
the isolates were collected in Beijing, China (14/35, 40%). The sources of these 35
132
isolates were sputum (20/35, 57.1%), blood (10/35, 28.6%), and abdominal fluid
133
(5/35, 14.3%). The general characteristics of the 35 A. baumannii genomes are listed
134
in Table S2.
135
Whole genome phylogeny of the genus Acinetobacter. To facilitate detailed
136
analysis of strain relationships, we developed a phylogeny based on single nucleotide
137
polymorphisms (SNPs) from whole genome alignment to represent the ancestral
138
relationships among 35 strains and 16 other A. baumannii strains (Fig. 1A). These
139
included 12 multidrug-resistant A. baumannii strains (AYE, AB0057, ACICU,
140
AB1656-2, TYTH-1, TCDC-AB0715, BJAB07104, BJAB0715, BJAB0868, ZW85-1,
141
MDR-TJ, and MDR-ZJ06), two susceptible strains (ATCC17978 and AB307-0294),
142
one community-acquired strain D1279779, and one non-clinical strain SDF isolated
143
from a human body louse. ADP1, a soil-living Acinetobacter baylyi strain was used as
144
the outgroup for comparison.
145
Based on the phylogenetic data, all the strains, along with nine previously
146
reported Asian strains, including MDR-ZJ06, MDR-TJ, ZW85-1, BJAB07104,
147
BJAB0715, and BJAB0868 from mainland China, TCDC-AB0715 and TYTH-1 from
148
China, Taiwan, and AB1656-2 from Korea, were grouped together with ACICU, a
149
strain of global clone II (GC II) group. The susceptible strains were clustered on the
150
edge of the evolutionary tree. Two groups of founder strains were identified among
151
the CRAB based on the phylogenetic tree, which were the earliest isolated strain
152
1999BJAB11 and two strains from Zhejiang isolated in 2004. Interestingly, the
153
earliest isolated strain 1999BJAB11 was separated from all of the other ST92 strains,
154
suggesting that it may have a different genome content, when compared with the other
155
ST92 strains.
156
The eBURST algorithm revealed one clonal complex (CC) CC1 and six
157
singletons (Fig. 1B). The CC1 contained ST92, which was identified as a potential
158
founder, with ST75, ST137, ST90, ST118, and ST138 radiating from it. All the
159
isolates in CC1 were CRAB, and the three susceptible strains were clustered into
160
singletons. The location and source of these isolates showed diversity in different ST.
161
Evolution of the resistance abaR islands. Ten types of the resistance abaR
162
islands were identified in the 32 CRAB strains (Fig. 2A). Except for the type 6 abaR
163
island, other types shared high homology. Type 10 abaR island was the predominant
164
type, which was identified in 12 strains (12/32, 37.5%), and was 19.123 kb in length
165
and contained the highest number of resistance genes, including tniB, usp, sul, tetA,
166
tetR, arsR, and aph, similar to those noted in a strain from Japan with one different
167
nucleotide (24). Type 5 abaR island in 1999BJAB11 contained the largest number
168
genes, and the conjugal transfer gene traA was identified only in this isolate. The
169
resistance genes tetA, tetR, arsR, aphA, and strB were not found in type 1 abaR island.
170
While
171
phosphoglucosamine mutase (glmM) were detected in type 2, 7, and 10 abaR islands.
172
The phosphoglucosamine mutase (glmM) can catalyze the conversion of
173
glucosamine-6-phosphate to glucosamine-1-phosphate, which is an essential step in
174
the formation of the cell wall precursor UDP-N-acetylglucosamine (25). In
175
Streptococcus gordonii, mutations in glmM appear to influence bacterial cell growth
176
and morphology, biofilm formation, and sensitivity to penicillins (26). Function of
177
glmM in A. baumannii was currently not well known. The mobile element protein was
178
not observed in type 3 and 10 abaR islands. Two strains from Zhejiang isolated in
179
2011 contained type 9 abaR island, which showed gene arrangement when compared
180
with type 8 abaR island. Two Zhejiang strains collected in 2004 had the type 6 abaR
181
island, which included the two-component response regulator, osmosensitive K+
182
channel histidine kinase KdpD, methyl viologen resistance protein smvA, RND
183
multidrug efflux transporter acriflavin resistance protein, and RND efflux system
184
membrane fusion protein CmeA. These genes were not observed in the other nine
the
sulfonamide
resistant
protein
dihydropteroate
synthase
and
185
types of abaR islands. The evolutionary ideograph of the abaR islands is shown in Fig.
186
2B, which was according to the time. The type 5 abaR island in the earliest strain
187
1999BJAB11 contained the largest number genes, and with the genome evolution,
188
gene acquirement, gene loss and gene rearrangement were detected in the strains
189
isolated during 2003-2011. The type 6 abaR island in two Zhejiang strains was
190
relatively independent of the other strains during abaR islands evolution.
191
Variations in transposon-associated antibiotic resistance genes. The
192
transposons containing drug resistance genes varied in different A. baumannii strains
193
(Fig. 3A). BlaOXA-51-like gene was located in three types of transposons. Eleven strains
194
possessed IS-blaOXA-51-like-IS transposon, and 10 of them were flanked by two ISAba1
195
elements. One strain 2011ZJAB2 was flanked by ISAba1 and ISAba16, and eight
196
strains possessed ISAba1-blaOXA-51-like-metallo-beta-lactamase-ISAba1 transposons.
197
Only
198
class-A-beta-lactamase-ISAba1 transposon. ISAba1-metallo-beta-lactamase-ISAba1
199
was detected in 11 stains, but in two strains from Zhejiang isolated in 2011,
200
metallo-beta-lactamase
201
ISAba1-class-A-beta-lactamase-ISAba1 was identified in 13 strains. In one strain
202
from Zhejiang isolated in 2004, ISAba20 and ISAba1 were detected on both the sides
203
of class A beta-lactamase; and in two strains from Guangdong isolated in 2011,
204
ISAba1 and ISAba13 were detected. In seven strains isolated in 2011, blaADC-30 was
205
located in ISAba1-blaADC-30-ISAba1, but in 2005LNAB4, blaADC-30 was flanked by
206
ISAba1 and ISAba18, and in two strains from Zhejiang isolated in 2011, blaADC-30 was
207
flanked by ISAba1 and ISAba14. Furthermore, in two strains from Zhejiang isolated
208
in 2004, ISAba1-ampC-ISAba1 was identified.
one
strain
2011LNAB2
was
flanked
possessed
by
ISAba14
ISAba1-blaOXA-51-like-
and
ISAba16.
209
The blaOXA-23-containing resistance island was identified in all the 32 CRAB
210
strains (Fig. 3B). In most of the sequenced strains (30/32, 93.8%), blaOXA-23 was
211
located in a transposon, which showed 99% identity to Tn2009. Tn2009 was initially
212
identified on the chromosome of A. baumannii MDR-ZJ06 from China, with a length
213
of 8421 bp and flanked by two ISAba1 elements. Tn2009 was noted to carry the
214
blaOXA-23 gene, the truncated DEAD/DEAH box helicase gene, the ATPase gene, the
215
yeeC gene, and the yeeB gene. However, in two isolates from Guangzhou obtained in
216
2011, blaOXA-23 was found in transposon Tn2006, which was shorter than Tn2009.
217
Tn2006 contained the ATPase gene, the yeeA gene, and the yeeB gene, and was
218
flanked by two ISAba1 elements.
219
Pan-genome: Core and accessory genes. All the sequenced A. baumannii
220
strains contained a genetically highly homogeneous core genome that encodes
221
proteins involved in DNA replication, transcription, and translation as well as many
222
metabolic pathways. By using the best reciprocal BLAST matches, we identified 2830
223
core genes/proteins and 1206 accessory genes/proteins among all the 35 A. baumannii
224
isolates. Graphing of the numbers of core and pan-genome as a function of the
225
number of strains sequenced revealed that the slope for the core gene number was
226
approaching an asymptote, whereas the pan-genome continued to expand even after
227
compilation of 35 genomes (Fig. 4A).
228
Variation in intrinsic chromosomal resistance genes related to carbapenem
229
resistance. The phylogenetic distribution of different alleles of blaOXA-51-like and
230
blaADC showed evidence of recombination and mutation. The presence of an upstream
231
insertion element, ISAba1, suggested that the entire region was replaced by
232
homologous recombination. The blaADC gene was always associated with upstream
233
ISAba1 oriented to allow overexpression of the gene, except in three susceptible
234
strains (Fig. 4B). The sequences from 19 strains were identical to the
235
extended-spectrum blaADC-30 variant (blue), and three strains showed 99% identity to
236
blaADC-30 (dark blue). Five strains contained blaADC-25 (yellow), and one strain carried
237
blaADC-56 (purple). One susceptible strain 2011BJAB9 and three resistant strains
238
showed 99% identity to blaADC-53 (orange), while blaADC in 1999BJAB1 showed 99%
239
identity to blaADC-29. The sequences from the susceptible strains 2011GDAB2 and
240
2011TJAB1 showed 98% and 99% identity to blaADC-52 and blaADC-63, respectively.
241
The blaOXA-51-like variants were different in the resistant strains and susceptible
242
strains. The most common blaOXA-51-like variant in resistant strains was blaOXA-66,
243
which was detected in 27 resistant strains (27/35, 77.1%), and blaOXA-68 were detected
244
in three resistant strains from Zhejiang. One unusual variant blaOXA-234 was detected
245
in a resistant strain from Jiangsu in 2005. Furthermore, three novel variants were
246
identified, including blaOXA-425 in the resistant strain 2011BJAB7 and blaOXA-424 and
247
blaOXA-426 in the susceptible strains 2011GDAB2 and 2011BJAB9, respectively. In the
248
susceptible strain 2011TJAB1, blaOXA-69 was detected. In most CRAB strains (25/32,
249
78%), blaOXA-51-like variants were associated with upstream ISAba1, but no upstream
250
ISAba1 was detected in all three susceptible strains.
251
IS-mediated T6SS and csuE region deletions. There were multiple instances of
252
chromosomal gene loss that might have possibly been mediated by an IS adjacent to
253
the deletion (Fig. 4C). Two large deletions adjacent to ISAba1 elements have been
254
previously described (10), including a 40-kbp region encoding the entire type VI
255
secretion system (T6SS) and a ~20-kbp region of adhesion genes (csuE) involved in
256
aspartate metabolism. In the present study, three different types of T6SS region were
257
noted, and eight strains lacked this region. Among these eight strains, in five strains,
258
deletion was mediated by IS insertion. It is worth noting that except for ISAba1
259
insertion in three strains isolated in 2011, ISAba13 insertion was detected in two
260
strains from Guangdong isolated in 2011. Similarly, three different types of csuE
261
region were also noted, and 12 strains lacked this region. Among these 12 strains,
262
deletion was mediated by IS insertion in three strains and transposase insertion in two
263
strains, respectively. Except for ISAba1-mediated deletion in 2011BJAB7, this study
264
is the first to identify ISAba125 insertion in two strains from Zhejiang isolated in
265
2004 and transposase insertion in the csuE region in two strains from Guangdong
266
isolated in 2011.
267
Variation in surface polysaccharide synthesis and heme utilization region.
268
The 35 strains showed substantial variation in the content and organization of loci
269
involved in surface polysaccharide synthesis (Fig. 4D). The core lipooligosaccharide
270
(LOS) loci (outer core [OC] locus) (27) consisted of one predominant type (blue),
271
which was present in most of the strains (29/35, 82.9%); however, we also identified
272
several variations in the three susceptible strains and one resistant strain. In two
273
strains from Zhejiang isolated in 2004, ISAba20 insertions were detected. With regard
274
to capsular (K locus) loci, 12 variations were identified. One predominant type (blue)
275
existed in 10 strains (28.6%), which was most closelgy related to ACICU (99.5%
276
identity at the nucleotide level).
277
The heme utilization region, containing several genes involved in heme
278
utilization, was more common in strains collected in 2011. However, this region was
279
absent in 1999BJAB1 and most of the strains collected during 2003–2005, except for
280
two strains from Zhejiang isolated in 2004. Among the 23 strains collected in 2011,
281
the heme utilization region was noted in 18 strains, which was 96% identical to
282
ACICU (Fig. 4E).
283
Variation in the restriction-modification system. The restriction-modification
284
(RM) system is a major participant in the co-evolutionary interaction between mobile
285
genetic elements (MGEs) and their hosts through the regulation of horizontal gene
286
transfer (HGT). In the present study, six strains clustered with two strains from
287
Zhejiang isolated in 2004 contained more RM system genes (Fig. 4F). In the
288
susceptible strain 2011TJAB1 and two resistant strains from Zhejiang isolated in 2004,
289
we identified Type I RM system methylase subunit, restriction subunit, and specificity
290
subunit. Furthermore, strains 2011ZJAB1 and 2011BJAB3 only had Type I RM
291
system methylase subunit, and strain 2011ZJAB4 only had Type I RM system
292
restriction subunit. Type II RM system methylase subunit was detected in strain
293
1999BJAB11 and susceptible strain 2011TJAB1, whereas Type III RM system
294
methylation subunit was found in three Zhejiang strains and two Guangdong strains.
295
CRISPR-associated protein was only detected in one susceptible strain 2011BJAB9.
296
Variation in phage-related regions. Phage-related regions are another source of
297
variability contributing to the difference among A. baumannii strains. Two
298
predominant types of phage-related regions were located within the same
299
chromosomal location corresponding to 1.11–1.16 Mbp in ACICU (ACICU_00997 to
300
ACICU_01077) and 1.45–1.53 Mbp in TYTH-1 (M3Q_1334 to M3Q_1458) (Fig.
301
4G). One primary variant typified by the completed reference genome of TYTH-1
302
consisted of two probable phage insertion events flanked by phage integrases (blue),
303
whereas another variant possessed only the second of the two phage elements (green).
304
Two susceptible strains 2011TJAB1 and 2011BJAB9 and strain 2011ZJAB4 had
305
different phage-related regions, when compared with the two main variants, and all
306
these strains were susceptible to tigecycline.
307
308
DISCUSSION
309
Despite increased research on A. baumannii evolution, the development of multidrug
310
resistance and genomic diversification of A. baumannii is not yet clear, especially in
311
China. In the present study, we have reported the whole genome sequences and
312
comparative genomic analysis of 35 representative A. baumannii strains isolated
313
during 1999–2011 in 13 hospitals in nine cities in China, including 32 CRAB and
314
three susceptible A. baumannii with different genotypes and phenotypes. These
315
genomes represent a significant addition of genomic data from the genus and could
316
serve as a valuable resource for future genomic studies.
317
It has been indicated that the resistance abaR islands have evolved into diverse
318
types through multiple events of insertion, deletion, and recombination (6, 28). The
319
predominance of type 4 abaR resistance islands among the CRAB isolates have been
320
reported in South Korea (29) and Taiwan (30). However, the evolution of resistance
321
abaR islands has not yet been investigated, especially in China. In the present study, it
322
was found that the type 6 abaR island in two Zhejiang strains collected in 2004 is
323
different from the previously identified abaR islands, which may thus be a novel abaR
324
island. The type 6 abaR island was noted to comprise the two-component response
325
regulator, osmosensitive K+ channel histidine kinase KdpD, methyl viologen
326
resistance protein smvA, RND multidrug efflux transporter acriflavin resistance
327
protein, and RND efflux system membrane fusion protein CmeA. These regulators
328
and resistance-related proteins in the type 6 abaR island may enhance the strain’s
329
adaptability and antibiotic resistance, which should be further studied.
330
Frequently encountered transposons and ISs carrying resistance genes in bacteria
331
reflect recombination flexibility during acquisition of resistance (4). In the present
332
study, each strain contained multiple transposons or ISs, which gradually tended to
333
diversify in the process of evolution. It must be noted that some of these ISs or
334
transposons are novel, and that most of the novel IS element insertion or transposons
335
were mainly found in strains from Zhejiang or Guangdong province of China. The
336
resistance genes or elements may spread through a transposon- or IS-mediated
337
mechanism, and the diversification of transposons or ISs in the process of evolution
338
increases the possibility of acquisition of novel resistance genes or elements.
339
Recombination has been reported to contribute to the change in the A. baumannii
340
genome (28). Furthermore, the heme utilization region is also highly variable among
341
the A. baumannii strains, which was more common in strains collected in 2011 in the
342
present study, thus making it difficult to assess whether this region was present in a
343
common ancestor and lost by some strains or was gained and subsequently transferred
344
via recombination to different lineages as initially observed. In addition, evidence of
345
recombination events in the allelic distribution of blaADC and blaOXA-51-like variants
346
was also noted. In all CRAB strains, the blaADC gene was associated with upstream
347
ISAba1 oriented to allow overexpression of the blaADC genes. However, in all three
348
susceptible strains, no upstream ISAba1 was detected. In addition, the blaOXA-51-like
349
variants were different in the resistant strains and susceptible strains. Four uncommon
350
blaOXA-51-like genes were identified, including blaOXA-234 and a novel blaOXA-type
351
blaOXA-425 in CRAB and novel blaOXA-type blaOXA-424 and blaOXA-426 in two
352
susceptible strains. In most CRAB strains (25/32, 78%), blaOXA-51-like variants were
353
associated with upstream ISAba1, but no upstream ISAba1 was detected in all three
354
susceptible strains. Furthermore, the blaOXA-23-containing resistance island was
355
identified in all the 32 CRAB strains, and absent in all susceptible strains. It is very
356
likely that blaOXA-234 or blaOXA-425 contributes to carbapenem resistance in A.
357
baumannii, as demonstrated previously with blaOXA-23, blaOXA-24, and blaOXA-58,
358
blaOXA-234 or blaOXA-425 was also associated with upstream ISAba1, which has been
359
shown to promote overexpression of OXA (31).
360
Despite extensive research on the virulence potential of A. baumannii, little is
361
known about its true pathogenic potential or virulence repertoire. A. baumannii
362
thrives in hospital settings largely due to its persistence on abiotic surfaces (32). One
363
mechanism for A. baumannii persistence in hospital settings is the presence of a
364
putative tip adhesion gene, csuE. The csuE gene is involved in pilus and biofilm
365
formation (33), and its presence has been associated with the persistence of A.
366
baumannii on abiotic surfaces such as plastic and glass (33). The resistant strains were
367
found to contain two different types of csuE. Most of the strains contained the
368
shortest-type csuE, which was not observed in 10 resistant strains. The second type of
369
csuE was only detected in two Zhejiang strains isolated in 2004, which comprised
370
ISAba125 and a gene cluster containing 16 proteins, when compared with the shortest
371
type. The T6SS secretion system is widespread among Gram-negative bacteria, and
372
can be used for toxicity against other bacteria and eukaryotic cells. The T6SS system
373
has been implicated in the interaction between bacteria as well as between bacteria
374
and their hosts. In Vibrio cholerae, the T6SS system confers toxicity toward other
375
bacteria, providing a means of interspecies competition to enhance environmental
376
survival (34). Furthermore, Pseudomonas aeruginosa has been found to activate the
377
T6SS system during infection in patients with cystic fibrosis (35), and also use the
378
T6SS-delivered toxins to actively kill competing bacteria (36, 37). In A. baumannii
379
strains, the T6SS secretion system is conserved and plays a role in competition with
380
other bacterial species (38). In the present study, three different types of T6SS region
381
were noted, with one type being predominantly detected. Two of the three susceptible
382
strains did not exhibit this region and only one strain presented a unique type of T6SS
383
region, which was different from those noted in the carbapenem-resistant strains. Two
384
resistant Zhejiang strains collected in 2004 exhibited the third type of T6SS region,
385
which contained more genes, when compared with the predominant type, including
386
VgrG, ATP synthase, and transcriptional regulators.
387
A. baumannii showed a strong ability to acquire foreign DNA such as drug
388
resistance and pathogenicity DNA, which allows the bacterium to acquire genetic
389
diversity and overcome antibiotic selection pressure. The flow of genetic information
390
between the bacterial cells by HGT drives bacterial evolution, and RM systems are
391
the key moderators of this process (39). The presence of DNA RM system may act as
392
a “selfish” genetic element to ensure its dissemination and/or may function in
393
bacteriophage defense and hindrance of lateral gene transfer (40). In the present study,
394
based on the phylogenetic tree, two groups of strains with different founder strains
395
were identified among CRAB. Furthermore, six strains clustered with two strains
396
from Zhejiang isolated in 2004 contained more number of RM system genes. The
397
transfer of resistance genes may facilitate the rapid spread of these genes among A.
398
baumannii strains, so the antimicrobial susceptibility profile of this pathogen should
399
be closely monitored. We speculate that new antibiotics with high ability to interfere
400
the horizontal transfer of the resistance genes or elements carrying them may
401
contribute to effective treatment of Acinetobacter infections in the future.
402
In conclusion, in the present study, we analyzed the genome of 35 representative
403
A. baumannii isolates from China. Extensive variation in the gene content was found,
404
even among strains that were phylogenetically and epidemiologically very closely
405
related. In the process of evolution, the genome of A. baumannii gradually tended to
406
diversify. Several mechanisms contributed to this diversity, including IS-mediated
407
deletions, genome-wide homologous recombination, transfer of mobile genetic
408
elements, and mobilization of transposons or ISs. Thus, the present study improves
409
our understanding of the evolutionary processes that contribute to the emergence of
410
CRAB in China. Future large scale sampling across different areas and time scales are
411
still needed to fully understand the evolution of A. baumannii and its drug resistant
412
development and trends
413
414
ACKNOWLEDGMENTS
415
This study was supported by the National Natural Science Foundation of China (No.
416
31170125), Beijing Natural Science Foundation (5122041), the Specialized Research
417
Fund for the Doctoral Program of Higher Education (20110001110043), and Key
418
Projects in the National Science & Technology Pillar Program (2012EP001002).
419
420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463
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Development
of a
multilocus
sequence typing
scheme for
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541
Figure legends:
542
Fig. 1. (A) Whole genome phylogeny of 35 A. baumannii and 17 sequenced A.
543
baumannii genomes. The phylogeny tree was constructed based on SNPs and rooted
544
with A. baylyi ADP1. Colored circles indicate different locations of the strains isolated.
545
Blue triangles indicate that the strains were susceptible to carbapenems. Predominant
546
MLST ST92 is highlighted in red. (B) Results of eBURST analysis conducted to
547
assign CCs in the 35 A. baumannii strains utilizing seven loci. The CCs are circled
548
and the predicted clonal ancestors are shown by the central points.
549
Fig 2. (A) Structure and distribution of the resistance abaR islands. (B) Evolutionary
550
ideograph of the abaR islands in the evolution of CRAB.
551
Fig 3. Transposons containing the drug resistance gene in different A. baumannii
552
strains. (A) Blue and green colors indicate ISAba1, orange color indicates ISAba1 and
553
ISAba16, yellow color indicates ISAba14 and ISAba16, gray color indicates ISAba20
554
and ISAba1, rose-red color indicates ISAba1 and ISAba13, pink color indicates
555
ISAba1 and ISAba18, purple color indicates ISAba1 and ISAba14, and blank indicates
556
the absence of these transposons containing the drug resistance gene. (B) Structures of
557
transposons containing blaOXA23, Tn2009-like (blue in A), and Tn2006 (green in A).
558
Fig 4. Genome features of the 35 A. baumannii strains. (A) Number of total features
559
in the core (green) and pan-(blue) genome as a function of the number of strains
560
sequenced. An average of 500 random permutations of the genome order is presented
561
for the pan- and core genome content; the error bars represent the standard deviation
562
of these results. (B) Intrinsic chromosomal-lactamase alleles. Different colors
563
represent different types; blue color indicates that blaADC is an ADC30 variant and
564
blaOXA66 variant. Horizontal bars indicate the presence of an upstream ISAba1. (C)
565
Variation in the presence of a T6SS gene cluster and csuE gene cluster. Different
566
colors represent different types, and blank indicates that the region is absent.
567
Horizontal bar represents ISAba1 or ISAba13 insertion locations within each locus. T
568
represents transposase insertion locations within each locus. (D) Surface
569
polysaccharide variants for the OC and capsular polysaccharide (K) loci. Different
570
colors represent different types. Horizontal bar represents ISAba1 insertion locations
571
within each locus. (E) Variant region showing recombination around the heme
572
utilization region. Orange color indicates that the heme region is present and blank
573
indicates that the region is absent. (F) RM system. Blue color indicates that the
574
subunit is present and blank indicates that the subunit is absent. (G) Phage content
575
refers to the phage-related region located in TYTH; blue color indicates that both the
576
phages are present, green color indicates that the first phage is present, and other
577
colors indicate that different phage is present (refer to the text for details).
578
