2 the multidrug-resistant environmental isolate E. coli ... · Page 6 of 52 95 drugs, with minimum...

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Insights into the environmental resistance gene pool from the genome sequence of 1

the multidrug-resistant environmental isolate E. coli SMS-3-5 2

3

4

W. Florian Fricke1, Meredith S. Wright

2,†, Angela H. Lindell

2, Derek M. Harkins

3, Craig 5

Baker-Austin2††

, Jacques Ravel1,*

and Ramunas Stepanauskas4 6

7

1 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, 8

MD 21201; 2

Savannah River Ecology Laboratory, University of Georgia, Aiken, SC 9

29802; 3

The J. Craig Venter Institute, Rockville, MD 20850; 4

Bigelow Laboratory for 10

Ocean Sciences, West Boothbay Harbor, ME 04575. 11

12

† Present address: Flathead Lake Biological Station, University of Montana, Polson, MT, 13

59860. 14

†† Present address: Centre for Environment Fisheries and Aquaculture Science, 15

Weymouth, Dorset DT4 8UB, UK. 16

17

* To whom correspondence should be addressed. E-mail: [email protected] 18

19

20

21

22

23

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00661-08 JB Accepts, published online ahead of print on 15 August 2008

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ABSTRACT 23

The increasing occurrence of multidrug resistant pathogens of clinical and 24

agricultural importance is a global public health concern. While antimicrobial use in 25

human and veterinary medicine is known to contribute to the dissemination of 26

antimicrobial resistance, the impact of microbial communities and mobile resistance 27

genes from the environment in this process is not well understood. Isolated from an 28

industrially polluted aquatic environment, E. coli SMS-3-5 is resistant to a record number 29

of antimicrobial compounds from all major classes, including two front-line 30

fluoroquinolones (ciprofloxacin and moxifloxacin) and in many cases at record-high 31

concentrations. To gain insights into antimicrobial resistance in environmental bacterial 32

populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome 33

sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 34

predicted to be involved in antimicrobial resistance were phenotypically characterized. 35

Using recombinant vector clones from shotgun sequencing libraries, resistance to 36

tetracycline, streptomycin and sulfonamide/trimethoprim was assigned to a single mosaic 37

region on a 130 kb plasmid (pSMS35_130). The remaining plasmid backbone shows 38

similarity to virulence plasmids from avian pathogenic E. coli (APEC) strains. Individual 39

resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial 40

isolates from multiple phylogenetic and geographic sources. Resistance to quinolones 41

was assigned to several chromosomal loci mostly encoding for transport systems that are 42

also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is 43

therefore dependant both on determinants acquired from a mobile gene pool likely 44

available to clinical and agricultural pathogens as well, and on specifically adapted 45

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multidrug efflux systems. The association of antimicrobial resistance along with APEC 46

virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence 47

through the extensive use of antibiotics. 48

49

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INTRODUCTION 49

Antibiotic resistance in bacterial pathogens is a worldwide major public health 50

challenge worldwide with increased morbidity and mortality due to bacterial infections 51

and associated costs in billions of dollars in the United States alone (48, 57). While 52

commensal bacteria are thought to function as reservoirs of antimicrobial resistance 53

genes for pathogens in host-associated communities (74, 77, 95), further research is 54

needed to address questions about the origin, presence and persistence of antimicrobial 55

resistance genes in the environment and their potential transfer from environmental to 56

clinical settings. 57

Most clinically relevant antibiotics originate from soil-dwelling actinomycetes 58

(43). Accordingly, microbial communities in the soil environment, where resistance is 59

necessary to counteract allelopathic compounds, have been shown to function as 60

resistance reservoirs to natural and artificial antimicrobials (20, 21, 70). Comparative 61

sequence analysis of different types of antimicrobial resistance genes suggests that they 62

originated and diversified in environmental communities, from which they were 63

mobilized and propagated into taxonomically and ecologically distant bacterial 64

populations (2). Plasmid exchange between environmental and host-associated bacterial 65

communities is a recognized source for the rapid spread of antimicrobial resistance 66

phenotypes (91). The potential significance of plasmids in disseminating antimicrobial 67

resistance genes is further accentuated by the association of plasmids with mobile genetic 68

elements such as transposons, integrons and IS elements (49, 88). To better understand 69

the evolution and dissemination of resistance phenotypes from clinical, agricultural and 70

environmental settings, it is therefore necessary to perform comparative sequence 71

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analysis of resistant isolates on three different levels, comparing whole genomes, single 72

plasmids and individual resistance gene cassettes. 73

The !-proteobacterium Escherichia coli is a ubiquitous member of the normal 74

gastrointestinal microflora of humans and animals (25). Since it is also widely present in 75

the environment, E. coli could function as a mediator for gene flow between 76

environmental and clinical settings. Highly adapted pathogenic E. coli have acquired 77

specific virulence attributes and successful combinations of these genetic factors persist 78

in E. coli pathotypes that cause disease in humans and agriculturally important animals 79

(42). Pathogenic E. coli fall into two main categories with intestinal isolates typically 80

causing enteric/diarrheal syndromes and extraintestinal isolates causing urinary tract 81

infections and sepsis/meningitis. The emergence of fluoroquinolone-resistant E. coli 82

strains from lineages associated with relatively lower virulence potentials (36) has led to 83

the hypothesis that the use of antibiotics could accelerate the evolution of formerly 84

commensal strains towards virulence (1). Previously sequenced E. coli genomes 85

originated mainly from pathogenic and host-associated isolates. In comparison to these 86

strains, the genome sequences of environmental E. coli isolates with antimicrobial 87

resistance phenotypes could provide insights into the evolutionary relationship between 88

antimicrobial resistance, the adaptation to pathogenic lifestyles and virulence. 89

E. coli SMS-3-5 was isolated from an industrial, metal-contaminated coastal 90

environment and was found resistant to a record-number of antimicrobials, in many cases 91

at record-high concentrations. Surprisingly, this environmental isolate is resistant to 92

ciprofloxacin and moxifloxacin, two front-line fluoroquinolones. In all, E. coli SMS-3-5 93

is resistant to six quinolone compounds, including first, second and third generation 94

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drugs, with minimum inhibitory concentrations (MICs) amongst the highest ever 95

recorded for E. coli (34). To our knowledge, E. coli SMS-3-5 is the first non-clinical E. 96

coli isolate found resistant to second and third generation quinolones. In the present 97

study, the genome of E. coli SMS-3-5 was sequenced and analyzed with emphasis on 98

both resistance and virulence-associated functions and compared to the genomes of seven 99

previously sequenced intestinal and extraintestinal E. coli strains as well as to the lab-100

adapted E. coli K12. In addition, recombinant vector clones from sequencing libraries 101

were used to phenotypically characterize selected antimicrobial resistance genes and 102

improve functional gene assignments. 103

104

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MATERIAL AND METHODS 104

Bacterial strain. E. coli strain SMS-3-5 was isolated from sediments of Shipyard 105

Creek, a tidal system in the harbor of Charleston, SC (32°50'32"N; 79°56'59"W), on 106

March 23, 2005. The creek drains the Macalloy Superfund Site and is heavily 107

contaminated with toxic metals and other industrial waste (94). After aseptic collection, 108

sediment samples were stored in sterile plastic bags on ice and processed within 24 hours. 109

Serial dilutions of sediment slurries were spread plated in triplicate onto 15 x 100 mm 110

petri dishes containing m-FC agar (Difco, Lawrence), following standard protocols for 111

fecal coliform counts in water (29). To select E. coli isolates, individual blue colonies 112

(fecal coliforms) were streaked to Nutrient Agar with 4-methylumbelliferyl-"-D 113

glucuronide (Difco Laboratories, 1998), incubated overnight at 37°C, and checked for 114

fluorescence under UV irradiation. Fluorescent isolates were considered to be E. coli. 115

PCR amplification and sequencing of the 16S rRNA gene was performed on a small 116

subset of isolates for unambiguous species verification. Positively identified isolates were 117

inoculated into cryovials containing 80% Tryptic Soy Broth and 20% glycerol. The 118

inoculated cryovials were coded to conceal their source, vortexed, and frozen (-80oC) for 119

later use in antimicrobial resistance screening assays. 120

Serotyping and ECOR grouping. Serotyping was performed at the Gastroenteric 121

Disease Center, Pennsylvania State University, PA. E. coli SMS-3-5 was classified 122

according to the ECOR system (32) by in silico analysis of the completed genome 123

sequence based on the rapid phylogenetic grouping PCR system technique (17). 124

Genome sequencing and annotation. Whole-genome random shotgun plasmid 125

insert libraries of 3-5 kbp and 10-12 kbp and fosmid insert libraries of 30-40 kbp were 126

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constructed as previously reported (68) and sequenced using 3730x1 DNA analyzer 127

(Applied Biosystems, Foster City). Recombinant plasmid vector clones were constructed 128

using the pUC-derived pHOS2 vector system (50) in combination with Thunderbolt™ 129

GC10™ Electrocompetent Cells (Sigma-Aldrich, St. Louis). Assembly and closure were 130

performed as previously described (68). 131

Comparative sequence analysis. Protein-encoding sequences (CDS) shared 132

between different chromosomes or plasmids were determined using the BLAST Score 133

Ratio analysis (67). In brief, for each predicted peptide sequence in a reference 134

chromosome the BLASTP raw score was collected for the alignment against itself 135

(REF_SCORE) and the most similar protein (QUE_SCORE) in each of the query 136

genomes analyzed. These scores were normalized by dividing the QUE_SCORE obtained 137

for each query plasmid protein by the REF_SCORE. In general, CDS with a normalized 138

ratio of >0.4 are identical over 30% of their full-length sequence. 139

Antimicrobial susceptibility testing. E. coli isolates from Shipyard Creek and 140

recombinant clones from the sequencing libraries were screened for susceptibility against 141

a panel of 26 antibiotics using microdilution onto commercial dehydrated 96-well 142

MicroScan! panels (Dade Behring, Sacramento, USA) in cation-adjusted Mueller-Hinton 143

broth (NCCLS 2004) according to the manufacturer’s instructions. The antimicrobials 144

present on the MicroScan! panels were chosen based on their mode of action, propensity 145

of resistance and clinical relevance. Tested antimicrobials from nine structural groups 146

included aminoglycosides: amikacin, 8-64 mg/L; gentamicin, 2-16 mg/L; streptomycin, 147

16–128 mg/L; apramycin, 8-32 mg/L; "-lactams: ampicillin, 4-32 mg/L; amoxicillin, 4-148

32 mg/L; penicillin, 16-128 mg/L; imipenem, 2-16 mg/L; meropenem, 2-16 mg/L; 149

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ceftriaxone, 8-64 mg/L; cefoxitin, 8-32 mg/L; cephalothin, 16-128 mg/L; cephalexin, 16-150

128 mg/L; folate synthesis inhibitors: trimethoprim, 2-16 mg/L; trimethoprim-151

sulfamethoxazole, 2/38-4/76 mg/L; sulfa agents: sulfathiazole, 250-500 mg/L; 152

nitrofurantoins: nitrofurantoin, 16-128 mg/L; tetracyclines: oxytetraycline, 4-32 mg/L; 153

tetracycline, 4-32 mg/L; quinolones: ciprofloxacin, 1-4 mg/L; moxifloxacin, 0.25-4 154

mg/L; nalidixic acid, 4-32 mg/L; ofloxacin, 1-8 mg/L; macrolides: azithromycin, 2-8 155

mg/L; erythromycin, 16-128 mg/L and chloramphenicols: chloramphenicol, 8-32 mg/L. 156

All concentration used in the analysis were successive doublings ranging from the 157

minimum to the maximum level. In addition to the antibiotics used on the MicroScan! 158

panels, resistance to the antimicrobial agents kanamycin, piperacillin, levofloxacin, 159

norfloxacin, sparfloxacin and rifampicin was also assessed using liquid media 160

microdilution as previously described (NCCLS 2004). For antibiotics with high-level 161

resistance successive doublings of antimicrobial concentrations were used until inhibition 162

was reached or until saturation of antimicrobials in liquid media over the course of the 163

susceptibility testing was evident. Thunderbolt™ GC10™ Electrocompetent Cells used 164

for the construction of plasmid vector sequencing libraries and a pHOS2 clone with an 165

recombinant insert spanning a 3.7 kbp region from the ribosomal protein gene cluster 166

served as negative controls for MIC tests. 167

Swarming motility assays. Swarming motility of E. coli SMS-3-5 was compared 168

to E. coli K12 and assayed using different growth conditions. Liquid cultures were 169

freshly inoculated from glycerol stocks and grown overnight in LB medium. Aliquots of 170

20-50 #l were dropped onto plates with 0.5%, 1.0%, 1.5% or 2.0% agar supplemented 171

with LB, BHI or marine broth medium (Difco). 172

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Accession numbers. The genome sequence of E. coli SMS-3-5 has been 173

deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank) under accession numbers 174

CP000970 (chromosome), CP000971 (pSMS35_130), CP000972 (pSMS35_8), 175

CP000973 (pSMS35_4) and CP000974 (pSMS35_3). 176

177

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RESULTS 177

The environmental E. coli isolate SMS-3-5. E. coli strain SMS-3-5 was isolated 178

from Shipyard Creek, an industrially contaminated tidal system in the Southeastern U. S. 179

The isolate was obtained as part of a large-scale coastal E. coli screening program and 180

was selected for in-depth analysis due to its exceptional level of antibiotic resistance. Of 181

433 isolates that were analyzed for this study 12% were resistant to 5 or more 182

antimicrobial compounds (C. Baker-Austin, unpublished data). E. coli SMS-3-5 is 183

tolerant or resistant to record-high numbers of antibiotics (32 out of 33 tested compounds 184

- Table 1) from eleven structural classes, with many of the minimum inhibitory 185

concentrations (MIC) above previously documented levels or at maximum drug 186

solubility. Extreme resistance was found to diverse aminoglycosides, penicillins, 187

cephalosporins, fluoroquinolones, macrolides, phenicoles, sulphonamides, and 188

tetracyclines. E. coli SMS-3-5 is resistant to exceptionally high concentrations of 189

nalidixic acid (>1,0000 #g/ml) the second-generation quinolones ciprofloxacin (200 190

#g/ml), norfloxacin (>500 #g/ml) and ofloxacin (>1,000 #g/ml), and the third-generation 191

quinolones levofloxacin (125 #g/ml) and moxifloxacin (45 #g/ml). To our knowledge, 192

the MIC for moxifloxacin is the highest and for ciprofloxacin the second highest on 193

record (34). According to the E. coli reference group (ECOR) typing system (32), E. coli 194

SMS-3-5 belongs to group D and its serotype is O19:H34. 195

196

The E. coli SMS-3-5 genome. The E. coli SMS-3-5 genome consists of five 197

replicons, a 5,068,389 bp chromosome and four plasmids (pSMS35_130: 130,440 bp; 198

pSMS35_8: 8,909 bp; pSMS35_4: 4,074 bp and pSMS35_3: 3,565 bp). General features 199

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of the E. coli SMS-3-5 compared to seven previously sequenced E. coli genomes are 200

shown in Table 2. The phylogenetic relationship of E. coli SMS-3-5 to 20 previously 201

sequenced E. coli and Shigella strains was investigated by multilocus sequence typing 202

(MLST) using the approach described by Wirth et al. (93). This MLST schema assigns E. 203

coli SMS-3-5 to a phylogenetic clade that is closer to the last common ancestor and 204

separates this isolate from any of the previously sequenced E. coli and Shigella strains 205

(Fig. 1). According to the comparison of E. coli SMS-3-5 to the MLST database of 462 206

E. coli isolates (web.mpiib-berlin.mpg.de/mlst) this strain belongs to the ST354 complex, 207

which contains several isolates from urinary tract infections (3 out of 5 isolates). 208

209

(i) The E. coli SMS-3-5 chromosome. Comparative sequence analysis was 210

carried out to identify unique genomic islands (GIs) and to further investigate the 211

evolutionary relationship of E. coli SMS-3-5 to seven previously sequenced E. coli 212

strains. The genome sequence of the nonpathogenic E. coli strain K12 served as reference 213

for this analysis (10). Other genome sequences included those of the avian pathogenic E. 214

coli strain APEC O1 (39), and of five human pathogenic E. coli strains: three 215

uropathogenic E. coli (UPEC) (UTI89 (13), CFT073 (90) and 536 (33)) and two 216

enterohaemorrhagic E. coli (EHEC) (O157:H7 EDL933 (65) and O157:H7 Sakai (30)). 217

Overall, the 5.06 Mbp chromosome of E. coli SMS-3-5 showed high sequence similarity 218

and gene synteny with these previously sequenced E. coli genomes (Fig. 2). Of the 4,743 219

CDS that are encoded on the E. coli SMS-3-5 chromosome, 341 CDS (7.2%) are unique 220

to this strain, whereas 3,230 CDS (68.1%) are shared with all seven previously sequenced 221

E. coli chromosomes (Supplementary Table S2). Unique features of the chromosome 222

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associate with GIs of deviating nucleotide composition patterns, IS elements and CDS 223

with divergent codon usages, indicating that these regions have been acquired through 224

horizontal gene transfer (Fig. 2). 225

On the E. coli SMS-3-5 chromosome, the distribution patterns of the genomic 226

islands that are shared by different subsets of previously sequenced E. coli chromosomes 227

confirm the close evolutionary relationship previously reported between APEC and 228

UPEC strains (39). They also indicate similarities in the presence or absence of genomic 229

islands between the two EHEC strains and K12 (Fig. 2). A large number of GI-encoded 230

CDS from the E. coli SMS-3-5 chromosome are shared with either the chromosomes of 231

the APEC and UPEC strains (104, 2.2%) or with the chromosomes of the EHEC strains 232

and E. coli K12 (133, 2.8%). For a complete list of conserved CDS see Supplementary 233

Table S2. Genomic islands shared between E. coli SMS-3-5 and UPEC/APEC tend to 234

encode virulence-associated functions (i.e., colicin, polysialic acid capsule biosynthesis, 235

and the general secretion pathway - Table 3). Whereas genomic islands shared between 236

E. coli SMS-3-5 and EHEC/K12 include a higher number of gene clusters involved in 237

metabolic functions (i.e., hydroxyphenylproprionate pathway, the first step of glutamate 238

fermentation (absent from K12), and the putrescine utilization pathway). The hyf operon 239

encoding for the biosynthesis of hydrogenase-4, absent from the APEC/UPEC strains, is 240

also present in E. coli SMS-3-5, the EHEC strains and K12. 241

Table 3 shows some of the GI-encoded CDS found in a subset of the other E. coli 242

genomes and directly implicated in virulence-associated functions. These GIs code, 243

among others, for the biosynthesis of the polysialic acid capsule (3), a cell invasion 244

system (35) and the heat-resistant agglutinin 1 (51). Ten chromosomal GIs are predicted 245

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to code for the biosynthesis of fimbrial (Fim1-7) or lipopolysaccharides (LPS) structures 246

that could be linked to virulence or could reflect adaptations of E. coli SMS-3-5 to its 247

environmental habitat. Except for a single fimbrial gene cluster, which is present in E. 248

coli SMS-3-5 and the UPEC strain CFT073 (Fim5), all other surface-related gene clusters 249

are either unique in E. coli SMS-3-5 (Fim1, Fim4-7, LPS1-3) or shared with the EHEC 250

strains and E. coli K12 (Fim2-3). 251

E. coli SMS-3-5 carries the complete Flag-2 gene cluster that encodes a novel 252

flagellar system and was first described in the enteroaggregative E. coli strain O42 (69). 253

This cluster is absent from all the other E. coli strains analyzed, except for the two 254

outermost genes lfhA and lafU (Supplementary Fig. S1). Flag-2 was shown to be present 255

in only 15 of the 72 ECOR strains tested by Ren et al. (69). In E. coli SMS-3-5, codon 256

usage or deviating nucleotide sequence analyses do not indicate integration of 257

horizontally transferred DNA at this chromosomal site making it likely that this cluster 258

was deleted in other E. coli genomes. The Flag-2 gene clusters from E. coli SMS-3-5 and 259

E. coli O42 are nearly identical (>94% nucleotide (nt) sequence identity) and display 260

similarities to the corresponding gene clusters from Aeromonas hydrophila (11) and 261

Vibrio parahaemolyticus (81) (Supplementary Fig. S1). Interestingly, the E. coli O42 262

Flag-2 gene cluster carries a single frameshift mutation in the chaperone protein lfgC, 263

which is consistent with the failure to elicit swarming motility in E. coli O42. While the 264

Flag-2 gene cluster appears to be intact and complete in E. coli SMS-3-5, we were not 265

able to reproducibly induce swarming motility in this strain (see Material and Methods). 266

The E. coli SMS-3-5 chromosome carries two unique metabolic islands that are 267

likely to increase the growth substrate spectrum of this strain compared to previously 268

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sequenced pathogenic E. coli strains. These islands code for sucrose (scrRABKY) and 269

raffinose (rafRABDY) uptake and degradation systems (Fig. 2). While rafRABDY was 270

previously described encoded on plasmids (5), in E. coli SMS-3-5 it is located within a 271

mosaic region of the chromosome containing the highest density of IS elements in the 272

entire genome. In this IS-rich region, E. coli SMS-3-5 also harbors CDS for the synthesis 273

and uptake of the siderophore aerobactin (iucABCD/iutA; (22)) and for an 274

iron/manganese transport system (sitABCD; (73)). Both transport systems have been 275

associated with virulence in APEC strains (72) where corresponding gene clusters are 276

located on virulence plasmids such as pAPEC-O1-ColBM (38) and pAPEC-O2-ColV 277

(41). A second copy of the sitABCD cluster in the E. coli SMS-3-5 genome is located on 278

the large plasmid pSMS35_130 (Fig. 3). Both clusters are highly conserved (95% nt 279

identity over the 3.5 kbp gene cluster), but similarity is limited to the sitABCD operon 280

and does not include the surrounding sequences. To our knowledge, this is the first time 281

that both the aerobactin and the iron/manganese transport system have been found 282

chromosomally encoded and that sitABCD is present in two different copies within the 283

same genome. 284

285

(ii) The E. coli SMS 3-5 plasmids. The largest plasmid from the E. coli SMS-3-5 286

genome, pSMS35_130, shares highly similar (>80% nt identity) sequence regions with 287

the two APEC virulence plasmids pAPEC-O1-ColBM ((38); 62% coverage) and pAPEC-288

O2-ColV ((41); 49% coverage) (Fig. 3, circle 1 and 2). In addition, pSMS35_130 shares a 289

subset of those regions with the resistance plasmids p1658/97 harbored by an E. coli 290

strain isolated from the human trachea ((98); 61% coverage) and pAPEC-O2-R ((40); 291

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47% coverage) from an APEC isolate (Fig. 3, circle 3 and 4). Based on the comparative 292

sequence analysis, pSMS35_130 can be divided into three regions that show distinct 293

degrees of conservation among resistance- and virulence-associated plasmids. These 294

encode functions related to plasmid transfer and maintenance, virulence and antibiotic 295

resistance (Fig. 3). 296

Antimicrobial resistance is encoded within a single continuous region on 297

pSMS35_130 (~42 kb) that is characterized by a high density of predicted horizontally 298

transferred DNA and a deviating nucleotide composition. Furthermore, a total of 32 299

transposase and integrase genes including ten copies of IS26 are spread across the 300

resistance region, in most cases marking the borders of syntenic regions shared between 301

pSMS35_130 and related plasmids (Fig. 3). The resistance region of pSMS35_130 shows 302

highest overall similarity to resistance plasmid pSC138 from Salmonella Choleraesuis 303

((16); >80% similarity, 64% coverage) and to a chromosomal genomic island from 304

Acinetobacter baumannii AYE ((26); >80% similarity, 53% coverage). In both cases, 305

syntenic stretches are shorter than 5 kbp and limited to the resistance region of 306

pSMS35_130. Individual resistance gene cassettes are composed of resistance genes and 307

adjacent mobile genetic elements and show widespread distribution among taxonomically 308

and biogeographically distinct drug resistant isolates (Table 4). The pSMS35_130 309

resistance region codes for resistance to chloramphenicol (cmlA), trimethoprim (dhfrV), 310

sulfonamides (sul2), aminoglycosides (strAB, aadA, aph), beta-lactams (blaT) and 311

tetracyclines (tetA). Sequencing clones harboring several of these genetic elements were 312

shown to confer antibiotic resistance to susceptible E. coli strains (Table 5). 313

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The remainder of pSMS35_130 is divided between a transfer (~54 kb) and a 314

virulence (~33 kb) region. pSMS35_130 transfer region has the most homogenous 315

nucleotide composition of the three regions, the lowest density of horizontally transferred 316

DNA and a strong bias in the orientation of the CDS (Fig. 3). It codes for type IV 317

secretion-related conjugative transfer (traABCDEFGHIJKLMNPQRSTUVWXY / 318

trbABCDEHIJ) and plasmid maintenance functions (hok, parB, sopAB, pemIK). 319

pSMS35_130 carries an incompatibility group IncFIIA origin of replication. The transfer 320

region accounts for most of the similarities observed between pSMS35_130 and the 321

antibiotic resistance plasmids pAPEC-O2-R and p1658/97 as well as the APEC virulence 322

plasmids pAPEC-O1-ColBM and pAPEC-O2-ColV (Fig. 3). pAPEC-O2-ColV can be 323

co-transferred with pAPEC-O2-R into plasmid-free laboratory E. coli strains (37). Since 324

both plasmids and pSMS35_130 harbor similar sets of CDS for F-type conjugative 325

transfer, it is likely that pSMS35_130 would be transferable to non-resistant strains. 326

However, due to the broad resistance spectrum of E. coli SMS 3-5 and the resulting lack 327

of counter-selection markers available, transferability of pSMS35_130 could not be 328

confirmed. Interestingly, it was not possible to cure pSMS35_130 from the E. coli SMS-329

3-5 genome using either novobiocin treatment (up to 100 #g/ml) or growth at 40°C. 330

The virulence-associated region of pSMS35_130 also shows a mosaic structure 331

with multiple inversions and loss of gene synteny when compared to the APEC virulence 332

plasmids pAPEC-O1-ColBM and pAPEC-O2-ColV or the antimicrobial resistance 333

plasmid p1658/97. The virulence region is absent from other antimicrobial resistance 334

plasmids and contains a subset of those virulence factors found on the APEC virulence 335

plasmids including the plasmid copy of the iron/manganese transport system sitABCD, an 336

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outer membrane protease gene (ompT), and a putative hemolysin F gene (hlyF). 337

pSMS35_130 encodes a colicin gene cluster that is similar to the one described on 338

pAPEC-O1-ColBM (38) but disrupted by insertions of IS1 and IS2. A total of five copies 339

of IS1 are present on pSMS35_130. These elements flank the transfer region, separate 340

some of the virulence genes in the virulence region, and are associated with breaking 341

gene synteny between pSMS35_130 and the other plasmids analyzed. The virulence 342

region carries a second origin of replication belonging to incompatibility group IncFIB. 343

Interestingly, the only other resistance plasmid with similarity to the virulence region 344

from pSMS35_130 lacks most of the putative APEC virulence factors (Fig. 3). 345

No evidence for the presence of additional resistance- or virulence-associated 346

functions is found on any of the three small plasmids pSMS35_8, pSMS35_4 and 347

pSMS35_3. pSMS35_8 shares a 6.5-kbp region (>90% nt identity) with ColE1 (12) 348

including the cea/imm gene cluster for colicin E biosynthesis and immunity. pSMS35_4 349

is almost identical (>74% nt identity) to the two cryptic E. coli plasmids pMG828-2 (99% 350

coverage; GenBank #DQ995352) and pIGWZ12 ((97); 96% coverage). No significant 351

sequence similarity could be found between pSMS35_3 and any known plasmid 352

sequence. 353

354

Antimicrobial resistance. To assess the phenotypic impact of selected loci from 355

the E. coli SMS-3-5 genome on the overall resistance phenotype of this strain, 356

antimicrobial resistance tests were carried out (Table 5) using sequencing clones carrying 357

genes thought to be involved in antimicrobial resistance. Minimum inhibitory 358

concentrations (MIC) of tested antimicrobial compounds were determined for the 359

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recombinant vector-containing clones and compared to the susceptible vector-less host 360

strain (Thunderbolt™ GC10™ Electrocompetent Cells, Sigma-Aldrich, St. Louis). A 361

clone that carried a recombinant vector insert with a fragment from the ribosomal protein 362

gene cluster served as an additional negative control. Based on the final genome 363

assembly, clones with short to medium insert sizes (3-12 kbp) were selected that 364

contained DNA spanning regions encoding for predicted antimicrobial resistance 365

functions from the chromosome or plasmid pSMS35-5_130. The tested loci included 366

CDS for known antimicrobial target proteins and for previously uncharacterized 367

multidrug efflux systems (Table 5). Due to the intrinsic ampicillin resistance markers 368

harbored by the plasmid cloning vector used to construct the sequencing library, we were 369

unable to assay for resistance to "-lactam antibiotics. However, expression of individual 370

and single copy sul2, dhfrV, cmlA, and tetRA genes in this cloning system conferred 371

antimicrobial resistance for sulfonamide, trimethoprim, chloramphenicol and tetracycline 372

to the susceptible E. coli host strain, respectively. Resistance levels of the recombinant 373

clones matched in all cases those established for E. coli SMS-3-5. In addition, the results 374

revealed that a sequence region encompassing genes for aminoglycoside 375

adenyltransferase (aadA) and a putative streptothricin acetyltransferase (sat-1) is able to 376

confer a sulfonamide resistance phenotype to the E. coli host strain that is equivalent to 377

the phenotypes observed for the recombinant sul2 clone and for E. coli SMS-3-5. AadA 378

and Sat-1 have been characterized to confer resistance to aminoglycosides (15) and 379

streptothricin (84), respectively, but not to sulfonamides. Further experiments are needed 380

to confirm these findings and explore the molecular mechanisms underlying the 381

resistance. 382

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383

Quinolone resistance. The most common and best understood mechanisms of 384

high-level quinolone resistance in E. coli involve mutations in the genes encoding for the 385

antimicrobial targets DNA gyrase (gyrA and gyrB) and DNA topoisomerase IV (parC 386

and parE) (34). Intrinsically involved with DNA replication, these genes are part of the 387

basic microbial housekeeping apparatus and are encoded on the chromosome. Single 388

amino acid substitutions in GyrA have been shown to suffice for high-level nalidixic acid 389

resistance in E. coli, but additional mutations in gyrAB or parCE are necessary for high-390

level fluoroquinolone resistance (31, 63, 86). Comparative sequence analysis of gyrAB 391

and parCE genes from E. coli SMS-3-5 with those of the quinolone-susceptible E. coli 392

strain K12 identified mutations that resulted in eleven amino acid substitutions, most of 393

which unique to E. coli SMS-3-5 (Fig. 4, Supplementary Table S3). The two amino acid 394

substitutions most commonly identified in GyrA from quinolone-resistant E. coli strains 395

(Ser83 and Asp87; (34)) are found in E. coli SMS-3-5. Expression of a recombinant 396

vector clone carrying a single copy of the E. coli SMS-3-5 gyrA gene in a quinolone-397

sensitive E. coli strain resulted in resistance to nalidixic acid (32 #g/ml, compared to 398

1024 #g/ml for E. coli SMS-3-5), but not in detectable resistance levels to ciprofloxacin 399

and ofloxacin (Table 5). Since the same clone also carries a CDS for a transporter of the 400

major facilitator (MFS) superfamily, which is absent from K12, the observed phenotype 401

could also be the result of a previously unknown transport mechanism. The genes gyrB, 402

parC and parE carry nucleotide polymorphisms that result in a total of nine amino acid 403

exchanges when compared to their counterparts in E. coli K12 (Fig. 4). To our 404

knowledge, only two of these, Ser80Ile and Glu84Gly in ParC, have been related to 405

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fluoroquinolone resistance in E. coli, where they confer resistance to ciprofloxacin in 406

combination with at least one mutation in gyrA (85). Due to the homology between the 407

subunits of DNA gyrase and DNA topoisomerase IV, amino acid substitutions in the 408

quinolone resistance-determining region in GyrA (Ser83 and Asp84) correspond to 409

substitutions of Ser80 and Glu84 in ParC (46). Of the nine additional substitutions found 410

in GyrAB and ParCE from E. coli SMS-3-5, four were also found in other protein 411

homologs from E. coli and Shigella (Supplementary Table S3). Interestingly, when 412

expressed individually in quinolone sensitive E. coli, parC, gyrB or parE did not induced 413

detectable quinolone resistance phenotypes, suggesting that a combination of these 414

mutations might be required to induce quinolone resistance phenotypes. 415

Alterations in two major regulons, the multiple antibiotic resistance operon 416

marRAB and the redox-sensitive regulatory operon soxRS, can decrease membrane 417

permeability and induce broad-range efflux pumps, thereby reducing microbial 418

susceptibility to various toxic compounds, including quinolones (18). In E. coli, 419

overexpression of marA has been shown to increase resistance to numerous 420

antimicrobials, but MICs determined for nalidixic acid (6.3 #g/ml), ofloxacin (0.1 421

#g/ml), and norfloxacin (0.1 #g/ml) (4) were low compared to E. coli SMS-3-5 (Table 1). 422

MarA acts as a translation repressor of the outer membrane porin OmpF, which is 423

believed to be critical in the diffusion of quinolones into the cell (18, 58). MIC testing 424

results show no increased resistance levels to quinolones for recombinant clones carrying 425

marRAB nor soxRS, whereas ompF-expression in a different clone could be responsible 426

for the observed ofloxacin-resistance phenotype (2 #g/ml) (Table 5). The lack of 427

resistance phenotypes for recombinant clones expressing marRAB or soxRS could result 428

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from dominant effects that copies of the same genes from the chromosome have on the 429

overall phenotypes of the host strain. ompF in E. coli SMS-3-5 carries several 430

insertion/deletions (indels) compared to the corresponding gene in E. coli K12. 431

Resistance could therefore result from altered OmpF properties or changes in the 432

expression of ompF. In addition to OmpF, MIC testing results associate three efflux 433

systems with ofloxacin resistance, including EefABC (4 #g/ml), a MFS transporter (2 434

#g/ml) with low similarity to NorA from Staphylococcus aureus (BAA14147; 25% 435

protein sequence identity), and Fsr (2 #g/ml). The outer membrane multidrug efflux 436

pump EefABC was identified in Enterobacter aerogenes and is functionally related to the 437

acriflavin resistance system AcrAB-TolC (53). While AcrAB-TolC together with MarA 438

was characterized in E. coli as responsible for a broad-range resistance phenotype to 439

tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampicin (61), eefABC 440

expression in E. aerogenes increases resistance levels to chloramphenicol, erythromycin 441

and ticarcillin, but not to quinolone compounds (52). In E. coli SMS-3-5, eefABC is part 442

of a large gene cluster that is completely absent in E. coli K12. Additional components 443

encoded in this gene cluster are a regulatory protein (EefR) and an additional membrane 444

transport protein (EefD). The norA-related gene from E. coli SMS-3-5 confers resistance 445

to ofloxacin alone, while norA from S. aureus confers resistance to both second-446

generation fluoroquinolones, ofloxacin and ciprofloxacin (96). Fsr is a distant member of 447

the major facilitator superfamily, suggesting that it may function as a proton-dependent 448

fosmidomycin efflux system (27, 60). According to our MIC testing results, fsr in E. coli 449

SMS-3-5, in addition to mediating resistance to ofloxacin, appears to also be involved in 450

ciprofloxacin resistance (2 #g/ml). A recombinant clone expressing the signal 451

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transduction protein gene pmrD and the MFS transporter gene yfbW also contributes to 452

ciprofloxacin resistance (2 #g/ml). While pmrD is known to induce polymyxin resistance 453

in some E. coli isolates (92), the function of yfbW is poorly understood. To our 454

knowledge, this is the first direct evidence for the involvement of fsr, and either pmrD or 455

yfbW in the resistance to quinolones and the first characterization of a new norA-related 456

quinolone resistance gene. Of all resistance factors that our MIC testing associated with 457

quinolone resistance, codon usage analysis predicts only the norA-related gene from the 458

E. coli SMS-3-5 chromosome to have originated from horizontal gene transfer. 459

460

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DISUSSION 460

The present study provides a new model for the functional characterization of 461

selected genes and gene clusters in whole-genome sequencing projects. Using 462

recombinant plasmid vector clones from shotgun sequencing libraries the impact of 463

specific genetic loci on the overall phenotypic characteristics of E. coli SMS-3-5 could be 464

demonstrated. Expression of genes encoded on sequencing plasmid vector inserts was 465

sufficient to detect changes in the susceptibility levels of a standard laboratory E. coli 466

strain. A methodological limitation to this approach is linked to the presence of the 467

antibiotic resistance marker gene on the plasmid sequencing vectors, which creates 468

background resistance. levels, and to the size of recombinant vector inserts, which 469

hinders the assignment of observed phenotypes to a single specific gene. 470

For over a dozen agents, encompassing several different structural classes of 471

drugs, E. coli SMS-3-5 demonstrated extreme antimicrobial resistance capabilities (Table 472

1), and in many cases up to saturation levels of a given test agent using broth 473

microdilution assays. Few other attempts have been made to ascertain absolute limits of 474

antibiotic resistance in E. coli using concentrations of individual agents that greatly 475

exceed established antimicrobial MIC breakpoints (i.e. CLSI guidelines). Stapleton et al. 476

(1995) (80) found high-level amoxicillin resistance with MIC levels at over 4000 µg/ml 477

in clinically-derived E. coli strains (E. coli SMS-3-5: >5000 µg/ml). Extreme resistance 478

to ampicillin (>400 µg/ml), chloramphenicol (>316 µg/ml) and tetracycline (>400 µg/ml) 479

was detected in fluoroquinolone resistant clinically-derived E. coli isolates (89), although 480

in most cases resistance levels were significantly lower than in E. coli SMS-3-5 481

(ampicillin: >2500 µg/ml; chloramphenicol: 256 µg/ml; tetracycline: >512 µg/ml). 482

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Resistance levels to oxytetracycline (#256 µg/ml) in agricultural E. coli isolates from 483

pigs and pig farmers were found comparable to those of E. coli SMS-3-5 (250 µg/ml) 484

(59). Mazzariol et al. (54) have detected significantly higher level of resistance to 485

ciprofloxacin in clinical E. coli isolates (>512 µg/ml) than determined for E. coli SMS-3-486

5 (200 µg/ml). To our knowledge, however, SMS-3-5 is the most fluoroquinolone 487

resistant environmental E. coli isolate studied to date. 488

The combination of whole-genome annotation and phenotypic characterization of 489

selected gene clusters revealed that most of the resistance potential of E. coli SMS-3-5, 490

including resistance to cephalosporin, tetracycline, chloramphenicol, sulfonamide, and 491

trimethoprim can be assigned to a ~42 kb resistance region on pSMS35_130. Among the 492

antimicrobial resistance phenotypes of E. coli SMS-3-5, high-level resistance to 493

quinolone compounds plays a particularly noteworthy role, since it is encoded solely on 494

the chromosome and in regions without indication of recent gene transfer, with the 495

exception of a norA-related gene for ofloxacin resistance. It was also demonstrated that 496

quinolone resistance most likely involves both known resistance mechanisms (i.e. 497

mutations in the quinolone target protein genes), and previously unknown or 498

uncharacterized resistance mechanisms (i.e. porins and efflux systems). Quinolone 499

resistance is mediated by combinations of E. coli housekeeping genes (gyrA and 500

potentially gyrB and parCE which contained a total of eleven amino acid substitutions) 501

and multidrug efflux systems that have for the most part, also been identified in E. coli 502

K12 and other sequenced E. coli isolates (fsr, eefRABCD, ompF, pmrD/yfbW). Together 503

with these transport systems MarA could contribute to the high level of quinolone 504

resistance. Resistance to different quinolone compounds is mediated by genes or gene 505

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clusters with specific, non-overlapping resistance spectra. On one hand, gyrA and/or the 506

adjacent MFS transporter gene induces resistance to the hydrophobic nalidixic acid but 507

not to the newer hydrophilic quinolones. On the other hand, the efflux systems OmpF, 508

EefABC, Fsr and the NorA-like protein confer resistance to second-generation 509

quinolones, but at relatively low levels and - except for Fsr - only to either ofloxacin or 510

ciprofloxacin. Moreover, the gyrA-containing sequence fragment is only responsible for a 511

fraction (32 #g/ml) of the total resistance phenotype to nalidixic acid (1024 #g/ml) 512

observed for E. coli SMS-3-5. These results demonstrate that resistance to different 513

quinolone compounds is decoupled and that the cooperative action of different 514

mechanisms contributes to the overall resistance phenotype. Furthermore, we 515

demonstrated the importance of efflux and membrane permeability factors in the high-516

level resistance to second-generation quinolones. 517

At least one study suggests a relatively high biological cost to fluoroquinolone 518

resistance in E. coli (8) implying that constant selective pressures are necessary to 519

maintain resistance in mixed populations. However, six out of 433 E. coli strains isolated 520

from Shipyard Creek expressed quinolone resistance (C. Baker-Austin, unpublished data) 521

showing that resistance in environmental E. coli populations at this site is rare but not 522

unusual. Quinolone residues have been shown to persist in surface waters (45). Shipyard 523

Creek is heavily contaminated with heavy metals such as chromium, copper, zinc and 524

strontium (94), and trace metals and other industrial waste at the isolation site may have 525

acted as indirect selection agents, e.g. by promoting microorganisms with effective 526

broad-range efflux pumps (7, 82). 527

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The physical origin of E. coli SMS-3-5 is unknown, making it difficult to predict 528

the selective conditions that triggered the evolution of its exceptional antimicrobial 529

resistance phenotype. Shipyard Creek is accessible for anthropogenic microbes from 530

multiple sources through coastal currents and tides, as it discharges straight into the 531

Charleston (SC) harbor. There are no known sewage sources in the Creek drainage area. 532

However, quinolones are widely used in cattle and poultry farming (64), in particular to 533

treat E. coli infections (78) and drug resistant bacteria from agriculture could leach into 534

the water through farming runoff. So far, APEC virulence plasmids have only been 535

identified in host-associated E. coli isolates. Even though nothing is known about the 536

prevalence of APEC-related resistance plasmids in the environment, the similarity of 537

pSMS35_130 to APEC virulence plasmids suggests a close association of E. coli SMS-3-538

5 with avian E. coli strains, one possibility being transport of E. coli SMS-3-5 to 539

Shipyard Creek by migrating birds. In any case, our study demonstrates that high-level 540

resistance to multiple frontline quinolones, which is mediated by multiple mechanisms, is 541

not limited to E. coli strains from clinical settings, but also present in environmental 542

populations. 543

The same site from which E. coli SMS-3-5 was isolated has also been sampled for 544

two other studies that addressed the prevalence of multidrug-resistant strains of Vibrio 545

vulnificus, a marine human pathogen without clinical reservoirs (6), and of mobile 546

genetic elements (94). In these studies, unexpectedly high numbers of multidrug-resistant 547

V. vulnificus strains were detected with 17.3% of all isolates being resistant to eight or 548

more antimicrobial compounds (6). However, while an increased abundance of class 1 549

integrons was found in samples from Shipyard Creek compared to non-contaminated 550

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sites (94), no consistent differences could be detected in the frequency of resistance 551

between V. vulnificus isolates from this site and pristine environmental estuaries (6). 552

MLST comparison separates the environmental strain E. coli SMS-3-5 from other 553

sequenced host-associated E. coli and Shigella isolates and suggests that it is more 554

closely related to their last common ancestor (Fig. 1). A possible explanation for these 555

findings would be that the genomes of host-associated E. coli and Shigella strains show 556

signs of adaptive evolution. Pathogenesis in E. coli is believed to result from the 557

acquisition and integration of horizontally transferred virulence factors into strain-558

specific chromosomal pathogenic islands or through the acquisition of plasmids 559

harboring these genetic factors (9, 23, 42). The comparative genome analysis of E. coli 560

SMS-3-5 with intra- and extraintestinal E. coli isolates provides clues that at least part of 561

the adaptation of previously sequenced E. coli strains to their host-associated lifestyles 562

was associated with genome reduction. The lateral flagellar gene cluster Flag-2 in E. coli 563

SMS-3-5, remnants of which are still present at conserved chromosomal sites in other E. 564

coli strains (69), provides an example for such a reductive evolutionary process. In this 565

context, a principal analogy has been suggested for pathogenic E. coli isolates and 566

laboratory strains such as E. coli K12, which has been propagated in nutrient-rich 567

cultures for many generations (28). The single polar flagellar operon on the E. coli K12 568

chromosome is a preferential target for genome decay providing substantial savings in 569

energy and amino acid requirements (19, 24). The same might hold true for the Flag-2 570

gene cluster in E. coli SMS-3-5. This cluster, which could be responsible for swarming 571

motility under specific, not yet characterized conditions, might represent a phenotypic 572

trait more advantageous to environmental E. coli populations. Further genome analysis of 573

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environmental and host-associated E. coli isolates is necessary to determine whether the 574

lateral flagellar cluster is in fact predominantly found in the environmental fraction of the 575

E. coli pan-genome. 576

The plasmid pSMS35_130 is a composite plasmid. IS elements IS26 and IS1 577

appear to have contributed to this mosaic structure, combining two separate regions 578

encoding for antimicrobial resistance and potential APEC virulence-related functions. 579

Limited to the resistance region of pSMS35_130, IS26 (ten copies) is part of or separates 580

individual conserved antimicrobial resistance gene cassettes. IS26 elements are widely 581

distributed among !-proteobacteria, where they are mostly associated with different 582

resistance genes (IS finder database - http://www-is.biotoul.fr/). Similar sequences have 583

also been observed in Gram-positive species (i.e. Corynebacterium; GenBank 584

#AM942444). A close association of IS26 with resistance genes in E. coli SMS-3-5 and 585

other bacterial isolates is likely to facilitate the horizontal spread of antibiotic resistance. 586

Repeated acquisition and integration events of individually acquired resistance gene 587

cassettes mediated essentially by recombination between IS26 or other repetitive 588

elements could therefore be the evolutionary drivers of the pSMS35_130 resistance 589

region. 590

Analogously, the virulence region of pSMS35_130 can be regarded as composed 591

of individual virulence gene cassettes that are separated from each other and from the 592

plasmid transfer backbone by five copies of IS1 (Fig. 3). The boundaries of such gene 593

clusters are apparent from interruptions in synteny between pSMS35_130 and the APEC 594

virulence plasmids pAPEC-O1-ColBM and pAPEC-O2-ColV. The two origins of 595

replication present in the transfer and virulence regions, respectively, suggest that 596

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pSMS35_130 evolved from an F-type ancestor through acquisition and integration of an 597

additional plasmid encoding for APEC-related virulence phenotypes. From an 598

evolutionary standpoint, the combination of transfer and virulence determinants on a 599

single plasmid would provide the selective benefit of a self-transferable virulence 600

phenotype. Additional APEC virulence factors and IS1 elements are also located on the 601

E. coli SMS-3-5 chromosome suggesting that recombinatorial exchange of conserved 602

virulence gene cassettes also involves the chromosome. However, the combined set of 603

virulence-associated genes from the chromosome and pSMS35_130 is reduced when 604

compared to related APEC plasmids (38, 41, 72). The lack of the salmochelin 605

biosynthesis operon (iroBCDEN) and the gene for increased serum survival (iss) could 606

therefore be indicative of a general tendency towards the decay of the APEC phenotype. 607

Altogether, the combination of the resistance region and the APEC-like virulence 608

region on plasmid pSMS35_130 has major implications for public health. Recently, such 609

APEC/resistance plasmid carrying a larger set of APEC virulence factors has been 610

identified in a poultry isolate of Salmonella enterica serovar Kentucky (W. F. Fricke, 611

unpublished data). Should more of these plasmids be promiscuous and transfer to other 612

strains or species, antibiotic use could play a critical role in co-selecting for multi-drug 613

resistance and virulence phenotypes. Moreover, APEC-related virulence genes have been 614

found up-regulated during UPEC infections in humans (79), including sitAB and iucAD, 615

both of which are present in E. coli SMS-3-5. Since it was demonstrated that a substantial 616

overlap between plasmid-associated virulence genes from APEC and UPEC strains exist 617

(71), it is possible to envision that APEC plasmids might function as a reservoir for 618

human UPEC virulence factors. 619

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The analysis of E. coli SMS-3-5 genome sequence clearly shows that large 620

fractions of antimicrobial resistance and potential virulence gene pools have been 621

acquired horizontally. These not only include the mosaic plasmid pSMS35_130 but also 622

chromosomal genomic islands, encoding among others for polysialic acid capsule 623

biosynthesis, the general secretion pathway, agglutinins, cell invasion and a putative type 624

III secretion systems. These genes have high similarity to genes found in other bacterial 625

isolates from diverse environmental and clinical settings. 626

Since the specific origin of the E. coli SMS-3-5 isolate remains obscure, it is 627

impossible to define the impact of the environment on its evolution or to determine to 628

which extent virulence and resistance determinants evolved in the environment. 629

Furthermore, because of the environmental origin of E. coli SMS-3-5, these gene pools 630

are likely to have been acquired without apparent selective pressure. In either case does 631

the comparative analysis of the E. coli SMS-3-5 genome presented in this study provide 632

evidence for the recent and efficient transfer of mobile virulence and resistance elements 633

between environmental and host-associated settings. This availability of a common 634

virulence and resistance gene pool to environmental bacteria as well as clinical pathogens 635

is of some concern and should be studied further. 636

637

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Acknowledgement. This work was supported with Federal funds from the National 637

Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, 638

Department of Health and Human Services, under NIAID Contract N01-AI-30071. 639

640

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969

970

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TABLE 1. Resistance levels of E. coli SMS-3-5. 971

Antibiotic MICa

[ug/ml] CLSI MICa breakpoints

[ug/ml]

ß-Lactams

Amoxicillin >5000 NDb

Ampicillin >2500 $32c

Cefoxitin 64 $32c

Ceftriaxone 32 $64c

Cephalexin 200 NDb

Cephalothin 64 $32c

Imipenem 2 $16c

Meropenem 2 $16c

Penicillin 250 NDb

Piperacillin >10000 $128c

Aminoglycosides

Amikacin 8 $64c

Apramycin 8 NDb

Gentamycin 2 $16c

Kanamycin >2500 $64c

Streptomycin >5000 NDb

Quinolones

Ciprofloxacin 200 $4c

Levofloxacin 125 $8c

Moxifloxacin 45 NDb

Nalidixic Acid >10000 $32c

Norfloxacin >500 $16c

Ofloxacin >1000 $8c

Sparfloxacin 125 NDb

Macrolides

Azithromycin 2 NDb

Erythromycin 16 NDb

Rifampicin 25 NDb

Sulfonamides

Sulfathiazole >5000 $512c

Trimethoprim >500 $16c

Trimethoprim/

Sulfamethoxazole

>500 $4/76c

Tetracyclines

Doxycycline 60 $16c

Oxytetracycline 250 NDb

Tetracycline >512 $16c

Miscellaneous

Chlorampenicol 256 $32c

Nitrofurantoin <16 $128c a Minimum inhibitory concentration. 972

b No current susceptibility guidelines according to NCCLS 2004 available. 973

c Agents for which established antimicrobial resistance guidelines are currently available. 974

according to NCCLS 2004 interpretive criteria for broth microdilution in Performance 975

Standards for Antimicrobial susceptibility Testing; Fourteenth Informational Supplement. 976

977

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TABLE 2. General features of the genomes of E. coli SMS-3-5 and seven previously 977

sequenced E. coli strains. 978

Strain Size GC

[%]

CDS

SMS-3-5

Chr 5,068,389 50 4,743

pSMS35_130 130,440 50 153

pSMS35_8 8,909 46 10

pSMS35_4 4,074 49 4

pSMS35_3 3,565 43 3

K12a

Chr 4,639,675 50 4131

EDL933b

Chr 5,528,445 50 5324

pO157 92,077 47 99

Sakaic

Chr 5,498,450 50 5253

pO157 92,721 47 85

pOSAK1 3,306 43 3

APEC O1d

Chr 5,082,025 50 4457

pAPEC-O1-R 241,342 47 225

pAPEC-O1-ColBM 174,242 50 198

UTI89e

Chr 5,065,741 50 5044

pUTI89 114,230 51 145

CFT073f

Chr 5,231,428 50 5379

536g

Chr 4,938,920 50 4629 a (10);

b (65);

c (30);

d (39);

e (30);

f (90);

g (33). 979

980

981

982

983

984

985

986

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TABLE 3. Putative virulence-associated functions encoded on the large resistance plasmid pSMS35_130 and in genomic islands of 1

the SMS-3-5 chromosome 2

Namea CDS Location Function Genes Distribution

b Best hitc

hlyF EcSMS35_A0098 pSMS hemolysin gene from avian E.

coli isolate (56) hlyF

APEC O1

(pAPEC-O1-ColBM) P1658/97 (pl.)

ompT EcSMS35_A0099 pSMS outer membrane protease gene

(72) ompT

APEC O1

(pAPEC-O1-ColBM) pAPEC-O2-ColV (pl.)

colB' EcSMS35_A0106-

A0107 pSMS

colicin-B biosynthesis and

immunity proteins (75, 76) cba', cbi'

d APEC O1

(pAPEC-O1-ColBM)

pAPEC-O1-ColBM

(pl.)

colM EcSMS35_A0111-

A0112 pSMS

colicin-M biosynthesis and

immunity proteins (44, 62) cma, cmi

APEC O1

(pAPEC-O1-ColBM)

pAPEC-O1-ColBM

(pl.)

Col EcSMS35_0116- 0121

Chr

uropathogen-specific colicin

biosynthesis and immunity

proteins

- UPEC, APEC O1 UTI89

Flag-2 EcSMS35_0241-

0284 Chr lateral flagellar operon (69)

lafABCDEFKSTUVWZ,

lfgABCDEFGHIJKLMN,

lfhAB, lfiEFGHIJMNPQR

- E. coli O42

sit /

sitABCD

EcSMS35_3170-

3173 (chr.)

EcSMS35_A0081-

A0084 (pSMS35)

pSMS/

Chr

iron/manganese transport system

gene clusters (73) sitABCD

APEC O1

(pAPEC-O1-ColBM) pAPEC-O2-ColV (pl.)

iut/iuc EcSMS35_3178-

3182 Chr

aerobactin biosynthesis and

transport system gene cluster (22) iucABCD, iutA

APEC O1

(pAPEC-O1-ColBM)

pAPEC-O1-ColBM

(pl.)

kps EcSMS35_3219-

3235 Chr

polysialic acid capsule

biosynthesis operon (3)

kpsCDEFMTUS,

neuABCDES APEC O1, UPEC UTI89

gsp EcSMS35_3238-

3248 Chr general secretion pathway operon gspCDEFGHIJKLM APEC O1, UTI89, 536 UTI89

TTS EcSMS35_4018-

4024 Chr

putative type III secretion system,

chaperone and effector

components

- - S. dysenteriae Sd197

(20%)e

HRA-1 EcSMS35_4755 Chr heat-resistant agglutinin 1 (51) UTI89, CFT073 E. coli O9:H10:K99

GimA EcSMS35_4856-

4869 Chr

cell invasion and carbon

metabolism (35)

ibeRAT, cglDECT,

gcxCIKR, ptnCKE APEC O1, UTI89 E. coli A0 34/86

Fim1 EcSMS35_0146-52 Chr putative fimbrial biosynthesis - (K12, APEC, UPEC)f -

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Fim2

Fim3

Fim4

Fim5

Fim6

Fim7

EcSMS35_0575-80

EcSMS35_2175-85

EcSMS35_2491-97

EcSMS35_2509-13

EcSMS35_3921-29

EcSMS35_4093-96

operons K12, EHEC

K12, O157:H7

-

CFT073

-

-

Sakai

Sakai

S. sonnei Ss046 (50%)

CFT073

-

S. flexneri 5b 8401

LPS1

LPS2

LPS3

EcSMS35_1020-28

EcSMS35_2255-68

EcSMS35_3959-67

Chr putative lipopolysaccharide

biosynthesis operons -

-

-

UPEC, APEC O1

-

-

536 a According to Fig. 2 and 3. 1 b Among completed E. coli strains (K12, EDL933, Sakai, APEC O1, UTI89, CFT073, 536) or pathotypes (UPEC: UTI89, CFT073, 536; EHEC: EDL933, Sakai). 2 c According to nucleotide sequence BLAST comparison with NCBI nr database. pl, plasmid. 3 d Truncated genes. 4 e Percentage of query region covered by best hit. 5 f Gene cluster with weak similarity present in non-EHEC strains. 6

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TABLE 4. Comparison of resistance gene cassettes from pSMS35_130 to the NCBI non-redundant nucleotide database 1

(www.ncbi.nlm.nih.gov) 2

Resistance

phenotypea

Gene compositionb

Length

[kb]

BLAST

Hits Bacterial isolate

b Accession Source of isolate

Cp IS26-cmlA-chp.-IS26 3.0 3

Salmonella sp. TC67, pl. TC67

K. pneumoniae NK245, pl. pK245

S. Choleraesuis SC-B67, pl pSC138

AB262968

DQ449578

AY509004

Japan (2006)d

Clinical isolate, Taiwan (2006)d

Septic patient, Taiwan (2002)

Tm int.-dhfrV 1.9 8


S. Typhi CT18, pl. pHCM1

Uncult. bacterium, pl. pRSB107

Achromobacter xylosoxidans

E. coli, pl. pHSH1

Aeromonas salmonicida, pl. pRAS1

DQ449578

AL513383

AJ851089

DQ393569

AY259085

AJ517790


Mekong river delta, Vietnam (1993)

Wastewater plant, Germany (2004)d

Cystic fibrosis patient, France (2006)d

Clinical isolate, China (2000-01)

Wild salmon, Norway (1989)

Su, Am IS5075-chp.-sul2-strAB 4.0 1 S. enteritidis, pl. pCAST2 AF542061 Korea (2002)d

Su, Am chp.-sul2-strAB 2.9 13

Vibrio cholera MO10, cTn SXT


S. Newport SL254, pl. pSN254

A. bestiarum 5S9, pl pAb5S9

S. Typhi CT18, pl. pHCM1

AY034138

DQ449578

CP000604

EF495198

AL513383

Clinical isolate, India (1992)


Retail meat, USA (2002)

River sediment, France (2007)d

Mekong river delta, Vietnam (1993)

ß-L blaT-tnpR'-IS26 2.3 1 S. Typhi CT18, pl. pHCM1 AL513383 Mekong river delta, Vietnam (1993)

ß-L blaT-tnpR' 1.3 11

K. pneumoniae 2412097A, pl. pRMH760

S. Parathyphi A AKU12601, pl. pAKU_1


S. Choleraesuis L2454, pl. pMAK1

E. coli 690FNR, pl. pLEW517

AY123253

AM412236

AJ851089

AB366440

DQ390455

Clinical isolate, Australia (1997)

Clinical isolate, Pakistan (2002)


Japan (2007)d

Primate feces, USA (1991-93)

Stt, Am sat-hyd.-aadA 3.0 8

S. Choleraesuis SC-B67, pl pSC138

E. coli

E. coli CVM1562, pl. pCVM1562

Enterobacter cloacae, pl. pQC

S. Typhimurium BF272

AY509004

EF560797

AY816216

DQ019420

EF051039

Septic patient, Taiwan (2002)

Pig caecum, Great Britain (2007)d

Diarrheic pig, USA (1998-99)

Clinical isolate, Netherlands (2002)

Portugal (2002-04)

T tnp.(Tn1721)'-chp.-

tetRA-trp.-chp.-tnp.' <6.2 6

S. Dublin L-789, pl. pMAK2

Uncult. bact., pl. pB10

E. coli JM109, Tn1721

S.Dublin, pl. pIE321

AB366441

AJ564903

X61367

EF633507

Japan (2007)d


Germany (1992)d

Spain (2007)d

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Aeromonas salmonicida, pl. pRAS1 AJ517790 Wild salmon, Norway (1995)

T (tnp.)-chp.-tetRA-trp.-

chp.-(tnp.') <5.7 8

Aeromonas caviae HG5B, pl. pRAS1

Acinetobacter baumannii AYE

Bordetella bronchiseptica, pl. pKBB4037

S.Typhimurium, pl. pFPTB1

E. coli, pl. pC15-1a

CR376602

CT025832

AJ877266

AJ634602

AY458016

Clinical isolate, Great Britain (1997)

France (2006)d

Germany (2005)d

Italy (2004)

Canada (2000-02)

Am IS26'-aphA-IS26 2.2 8

Pseudomonas aeruginosa 2293E

S. Typhimurium G8430, pl. pU302L


Shigella flexneri SH595, TnSF1

pl. NTP16

L36547

AY333434

AJ851089

AF188331

L05392

(1994)d

Clinical isolate, USA (2006)d


Taiwan (1999)d

Great Britain (1992)d

- IS26 0.8 108 !-proteobacteriae

Corynebacterium sp. (Actinobacteria) n/a n/a

a Antibiotics: Cp, chloramphenicol; Tm, trimethoprim; Su, sulfonamide; Am, aminoglycoside; ß-L, beta-lactam; Stt, streptothricin; T, tetracycline. 1 b Abbreviations: chp, conserved hypothetical protein; int, integrase/recombinase; hyd, hydrolase; tnp, transposase; trp, transporter. Gene names in italics. 2 Truncated elements are marked with >'<. Genes in parentheses are not present in all hits. 3 c Only continuous regions showing gene synteny and high nucleotide sequence identity (>90%) are shown as individual hits. pl., plasmid; cTn, conjugative 4 transposon. 5 d Estimated year of isolation based on submission of publication. 6 e Similar IS26 elements (100% coverage, >99% nt identity) have been identified in species of the following geni (alphabetical): Acinetobacter, Citrobacter, 7 Enterobacter, Escherichia, Klebsiella, Pasteurella, Photobacterium, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, Yersinia. 8 9

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TABLE 5. Resistance profiles of E. coli SMS-3-5, the laboratory E. coli strain EPI-300 without plasmid vector, and selected 1

recombinant plasmid vector clones of EPI-300 from the whole-genome shotgun sequencing library of SMS-3-5 2

Strain/

Clone Clone inserts

a

Distributionb

[Coverage in %]

Minimum inhibitory concentrationsc

[!g/ml]

Size

[kbp] CDS Genes

d Rep K12 Other E. coli N Cf O Ot T St Tm / Sm Cp

SMS-3-5 - - - - - - 32 (1024) 4 8 32 32 (512) 500 (>5000) 4/76 (>500, >500e) 32 (2

GC10f - - - - - - 0 0 0 0 0 0 0 0

EC1BG86 - - - - - - 0 0 0 0 0 0 0 0

EC2A028 9.6 11 dhfrV, cmlA, IS26 Pl 0 0 (13-23) 0 0 0 0 0 0 4/76 (>500,76e) 32 (2

EC1DP02 3.2 3 sul2, IS5075 Pl 0 0-2 (0-6) 0 0 0 0 0 500 (500) 0 0

EC1AK08 4.1 4 aadA, sat-1, IS26 Pl 0 0 (0-59) 0 0 0 0 0 500 (500) 0 0

EC1BD74 4.4 5 tetRA, IS26, tnp-Tn1721 Pl 0 0 (0-55) 0 0 0 32 32 (512) 0 0 0

EC2AN65 10.0 7 fsr Chr 99 98-99 0 2 2 0 0 0 0 0

EC2A927 12.0 9 (eefRABCD) Chr 34 99-100 0 0 4 0 0 0 0 0

EC2CI57 9.3 7 ompF Chr 100 100 0 0 2 0 0 0 0 0

EC2AW38 11.6 7 gyrA (MFS, GntR.) Chr 66 66-67 32 (32) 0 0 0 0 0 0 0

EC2CO68 gyrBg Chr 0 0 0 0 0 0 0 0

EC2AN16 9.3 7 (MFS~nor, LysR.) Chr 75 75-99 [EHEC] 0 0 2 0 0 0 0 0

EC2AC67 11.2 10 pmrD, yfbW Chr 100 100 0 2 0 0 0 0 0 0a A complete list of all clone inserts with corresponding CDS and coordinates is part of the supplement (Table S4). 3 b According to BLAST comparison. Other E. coli: O157:H7 EDL, Sakai, APEC O1, UTI89, CFT053, 536. Fractions present on plasmids in parentheses. 4 c As determined on 96-well MicroScan! Panels. MIC values for individual clones and antimicrobials as determined using liquid media microdilution in 5 parentheses. Antimicrobial compounds: N, nalidixic acid (quinolone); Cf, ciprofloxacin (fluoroquinolone); O, ofloxacin (fluoroquinolone); Ot, oxytetracycline 6 (tetracycline); T, tetracycline; St, sulfathiazole (sulfonamide); Tm/Sm, trimethoprim/sulfamethoxazole; Cp, chloramphenicol. 7 d Gene names (in italics) and abbreviations: fsr, fosmidomycin resistance protein; CPA-2, monovalent cation:proton antiporter-2; Bcr/Acr, multidrug resistance 8 gene cluster with similarity to bicyclomycin and acriflavin resistance proteins; ompF, outer membrane protein F; gyrA, DNA gyrase subunit A; MFS, transporter 9 of the major facilitator superfamily; GntR., transcriptional regulator, GntR family; MFS~nor, transporter with weak similarity (25% amino acid identity) to 10 quinolone resistance protein NorA from St. aureus; LysR., transcriptional regulator, LysR family; pmrD, polymyxin B resistance protein; yfbW, small multidrug 11 resistance family protein. Genes not present in K12 are shown in parentheses. 12 e MIC determination for trimethoprim and sulfamethoxazole in liquid media were performed separately. 13 f Thunderbolt™ GC10™ Electrocompetent Cells (Sigma-Aldrich) and a sequencing clone (EC1BG86) spanning a region from the ribosomal protein gene cluster 14 were used as negative controls. 15

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g Other clones without resistance phenotype to any of the antibiotics listed carried inserts with parC-E, soxRS, mdtABC-D-EF-G-H-IJ-K-L-M-NOP, acrRAB-EF, 1 marRAC, envRAB, mdlAB, macAB, mdfA. 2

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FIGURE LEGENDS 1

Figure 1. Maximum likelihood phylogenetic tree of sequenced Escherichia and Shigella 2

strains based on multilocus sequence typing (MLST). Alignments were produced with 3

ClustalW (14). Modeltest 3.5 (66) was used to determine the most appropriate 4

evolutionary model. The tree was created with PAUP (83). Nodes marked with circles 5

have !70% neighbor-joining bootstrap support. Similar results were obtained for trees 6

calculated with neighbor-joining and parsimony methods. 7

8

Figure 2. Circular representation of the E. coli SMS-3-5 chromosome. Circles from 9

outside to inside: Circles 1+2, strand-dependent depiction of all CDS. Circle 3, IS 10

elements (red) and laterally transferred CDS (dark green), predicted with SigiHMM (87). 11

Circle 4, trinucleotide sequence composition; Circles 5-11, NUCmer (47) sequence 12

comparisons (>95% nt identity) with the chromosomes of E. coli strains K12 (circle 5), 13

O157:H7 EDL933 (circle 6), O157:H7 Sakai (circle 7), APEC O1 (circle 8), UTI89 14

(circle 9), CFT073 (circle 10), and 536 (circle 11). Sequence matches in the same 15

orientation are shown in red, in inverted orientation in blue. Circle 12, cumulative GC 16

skew. Selected GIs on the SMS-3-5 chromosome are highlighted depending on functional 17

annotations in light green (virulence factors - Table 3), gold (surface structures), orange 18

(phages), blue (metabolism), and grey (unknown functions). Genetic loci involved in 19

quinolone resistance are marked with black arrow heads with designations corresponding 20

to Table 5. Abbreviations (clockwise, genes names in italic): Col, colicin; Fim1-7, 21

fimbriae; mhpRABCDEF, 3-hydroxyphenylpropionate degradation; Glu, glutamate 22

fermentation (mutE, mamS); LPS1-3, lipopolysaccharide biosynthesis; lsrRABCDFGK, 23

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LuxS-regulated autoinducer uptake and modification; puuRABCD, putrescine utilization; 1

hyfABCDEFGHI, hydrogenase-4; cas, CRISPR-associated proteins; scrRABKY, sucrose 2

uptake and degradation; rafRABDY, raffinose uptake and utilization; sitABCD, 3

iron/manganese transport; iucABCD/iutA, aerobactin biosynthesis and transport; 4

kpsCDEFMTUS/neuABCDES, polysialic acid capsule biosynthesis; gspCDEFGHIJKLM, 5

general secretion pathway; glcABCDEFG, glycolate utilization; PTS, carbohydrate-6

specific PTS system; TTS, Salmonella type III secretion system (sicA, sipBD, hilA); 7

alsRABCEK, allose uptake and utilization; HRA-1, heat resistant agglutinin 1; GimA, 8

carbon source-regulated cell invasion (35). 9

10

Figure 3. Circular representation of pSMS35_130. pSMS35_130 is shown as composed 11

of a transfer, virulence and resistance region. Circles from outside to inside: Circles 1+2 12

(from different regions), strand-specific depiction of all CDS, color-coded according to 13

predicted function in grey (replication), dark green (transfer and maintenance), 14

brown/gold/red (transposition and recombination), light blue (APEC virulence), pink 15

(colicin biosynthesis and immunity) and green (antibiotic resistance). IS1 elements are 16

depicted in gold, IS26 elements in red, all other CDS in black. Circles 3-6, NUCmer 17

sequence comparisons (>90% nt identity) with the E. coli plasmids p1658/97 (circle 3), 18

pAPEC-O1-ColBM (circle 4), pAPEC-O2-ColV (circle 4), pAPEC-O2-R (circle 6), and 19

pSC138 from Salmonella enterica serovar Choleraesuis (circle 6). Sequence matches in 20

the same orientation are shown in red, in inverted orientation in blue. Circle 7, laterally 21

transferred CDS (black). Circle 8, trinucleotide sequence composition. Abbreviations 22

(clockwise, gene names in italics): tra/trb (traABCDEFGHIJKLMNPQRSTUVWXY, 23

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trbABCDEHIJ), type IV secretion system-like conjugative transfer system; parB', ParB-1

like partition protein; ssb, single-strand binding protein; sitABCD, iron/manganese 2

transport system; ompT, outer membrane protease 7; colB', truncated colicin-B 3

biosynthesis and immunity proteins; colM, colicin-M biosynthesis and immunity 4

proteins; cmlA, chloramphenicol transferase 2; dhfrV, dihydrofolate reductase type V; 5

sul2, dihydropteroate synthase type 2; strAB, aminoglycoside resistance proteins A and 6

B; blaT, beta-lactamase TEM; aadA, aminoglycoside adenylyltransferase; tetRA, 7

tetracycline resistance repressor and resistance protein, class A; aph, aminoglycoside 3’-8

phosphotransferase. 9

10

Figure 4. Mutations in the subunits of DNA gyrase (gyrAB) and DNA topoisomerase IV 11

(parCE) encoded on the E. coli SMS-3-5 chromosome compared to E. coli K12. Arrows 12

indicate resulting amino acid substitutions from E. coli K12 to E. coli SMS-3-5. 13

Chromosomal coordinates of the genes are shown. Amino substitutions known to be 14

involved in quinolone resistance (34) are shaded in grey. Novel and unique amino acid 15

substitutions are marked with boxes. 16

17

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IHE3034

F11

101-1

B7A

E110019

B171

E22

ATCC 8739

E. albertii TW07627

(outgroup)

0.01

APEC 01

UTI89

CFT073

536

E24377A

Shigella sonnei Ss046

K12

53638

HS

Shigella boydii Sb227

O157:H7 EDL933

O157:H7 Sakai

Shigella dysenteriae Sd197

SMS-3-5

Figure 1

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1.5

0.5

fsr

1.0

2.0

2.5

3.0

3.5

4.5

0Flag-2

mhp

Fim1Col

LPS1

lsr

puu

Fim3

LPS2

Fim4Fim5hyf

casscr

raf

iuc/iut, sit

glc

kpsgsp

Fim6LPS3

PTSTTS

Fim7

GimA

als

HRA-1

Glu

Fim2

eef

omp

gyrnorpmr

ori

Figure 2

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ResistanceIS26

IS26

IS26IS26

Transfer

Virulence

IS26

IS26

IS26

IS26

IS26

IS1

IS1

IS1

IS1

IS1

IS26

cmlA

dhfrV

sul2strABblaT

aadA

tetRA

aph

colM

colB’

IncFIB

sitABCD

ompThlyF

parB’ssb

repA1

repA2

tra

/ trb

IncFIIA

Figure 3

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gyrA gyrBparC parE

S83L

D87N

S80I

E84G

D475E

V136I

V153I

I355T

E185D

S492N

A618T

2,420,390

2,423,017

3,386,493

3,388,751

3,402,026

3,403,918

4,154,373

4,156,787Chr.

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