Journal of Antimicrobial Chemotherapyirep.ntu.ac.uk/id/eprint/6481/1/205594_7750 Mcnally Postprint.pdf · Journal of Antimicrobial Chemotherapy: under review Journal of Antimicrobial

Confidential: for peer review only

Molecular epidemiology of Extra-Intestinal Pathogenic E.

coli isolates from a regional cohort of elderly patients highlights prevalence of ST131 strains containing increased antimicrobial resistance in both community and hospital

care settings

Journal: Journal of Antimicrobial Chemotherapy

Manuscript ID: Draft

Manuscript Type: Original Article

Date Submitted by the Author:

n/a

Complete List of Authors: Croxall, Gemma; Nottingham Trent University, Pathogen Research Group Hale, James; University College Cork, Environmental Research Institute Weston, Vivienne; Nottingham University Hospitals, Microbiology Manning, Georgina; Nottingham Trent University, Pathogen Research Group Cheetham, Philip; Nottingham Trent University, Pathogen Research Group

Achtman, Mark; University College Cork, Environmental Research Institute McNally, Alan; Nottingham Trent University, Pathogen Research Group

Keywords: Escherichia coli, ESBLs, Uropathogenic strains, Urinary tract infection (UTI), Virulence genes

Journal of Antimicrobial Chemotherapy: under review

Journal of Antimicrobial Chemotherapy


Molecular epidemiology of Extra-Intestinal Pathogenic E. coli isolates from a regional 1

cohort of elderly patients highlights prevalence of ST131 strains containing increased 2

antimicrobial resistance in both community and hospital care settings 3

4

Gemma Croxall1, James Hale

2, Vivienne Weston

3, Georgina Manning

1, Phil Cheetham

1, 5

Mark Achtman2 and Alan McNally

1* 6

7

1Pathogen Research Group, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8

8NS 9

2 Environmental Research Institute, University College Cork, Lee Road, Cork, Ireland. 10

3Nottingham University Hospitals, Nottingham, England. 11

12

13

14

15

16

17

*Corresponding author: Dr Alan McNally, Pathogen Research Group, School of Science and 18

Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS. 19

Tel: 0044 +115 8483324 20

Email: [email protected] 21

22

23

24

25

26

Page 1 of 18




Abstract 27

Objectives 28

To assess the molecular epidemiology and prevalence of antibiotic resistance in E. coli 29

causing urinary tract infections of elderly patients from community and hospital settings. Also 30

to determine whether the possession of antibiotic resistance and virulence associated genes 31

can be linked to patient location or clonal group of the organisms in question. 32

Methods 33

E. coli were isolated from the urine samples of elderly UTI from the Nottingham area and 34

subjected to antibiotic susceptibility testing, virulence gene detection by PCR and MLST 35

analysis. 36

Results 37

No correlation was observed between community or hospital derived strains with regards to 38

antibiotic resistance levels or virulence gene profiles. E. coli ST131 was the predominant 39

sequence type found in both hospital and community samples and demonstrated high levels 40

of antibiotic resistance to the test panel but did not possess a significantly larger array of 41

virulence genes, nor a specific gene profile compared to other sequence types. 42

Conclusions 43

The level of antibiotic resistance or virulence gene possession in uropathogenic E. coli is not 44

directly associated with the health care setting of the patient, but there is a variation in 45

antibiotic resistance and virulence gene possession depending on clonal group. ST131 is 46

highly virulent and demonstrates high levels of antibiotic resistance but its virulence does not 47

appear to be attributed to the possession of a specific virulence associated gene set, nor the 48

possession of any VAG in significantly higher levels than any other sequence type. 49

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Introduction 50

Urinary tract infection is one of the most common nosocomial infections in the UK and also 51

has a significant occurrence in the community.1,2

It is well known that E. coli is the primary 52

etiologic agent of UTI in all age ranges, accounting for up to 80% of infections.3,4

Although 53

once considered to be a clonal organism, it is now widely accepted that E. coli as a species is 54

extremely heterogeneous and is subject to high rates of recombination within the accessory 55

genome.5

Due to this high level of heterogeneity in the species, E. coli are broadly 56

categorized into three main groups; (1) commensal E. coli, (2) intestinal E. coli such as ETEC, 57

EPEC, EHEC and (3) Extra-intestinal Pathogenic E. coli or ExPEC. From this level of 58

classification E. coli are usually further sub-divided into pathotypes on the basis of isolation 59

site and the possession of certain “virulence factors”. For example within ExPEC, E. coli 60

causing UTI are termed UPEC or uropathogenic E. coli on the basis that they were isolated 61

from the bladder and possess at least two UPEC associated virulence factors.6 Although it is 62

now appreciated that the level of heterogeneity within this species is immense, in a clinical 63

setting UPEC are still traditionally classed and treated as a homogenous group of organisms. 64

Due to the era of multilocus sequence typing we now know that E. coli strains within a 65

population can vary greatly in terms of their evolutionary descent, which in turn can affect 66

pathogenic potential and fitness in an infection. This is especially so in the case of the 67

recently emerged ST131, which has been linked to community acquisition, and more 68

significantly the world-wide dissemination of CTX-M-15.7-11

69

70

It has been well reported that a reservoir of ESBL-producing organisms that usually cause 71

urinary tract infections exists in long-term care facilities such as nursing homes.7,12

This will 72

inevitably pose a greater risk to elderly patients living within long-term care facilities. Previous 73

findings by our group reported that significant variations exist within a population of E. coli 74

causing UTI in the Nottingham area and that a subgroup of organisms with increased 75

virulence potential and antimicrobial resistance existed within this population.13

This 76

investigation aimed to compare the molecular epidemiology, antimicrobial activity and 77

virulence-associated gene (VAG) carriage of the aforementioned E. coli isolated from two 78

populations within the same geographical area, namely the community and the hospital 79

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setting and to determine if any observed differences could be attributed to clonal group or the 80

possession of a specific virulence associated gene set. 81

82

Methods 83

84

Bacterial isolates 85

Two hundred and fifty urine culture plates were collected at random from Nottingham 86

University Hospitals (NUH) between October 2008 and June 2009. Cultures were collected 87

anonymously (therefore no ethical approval or informed consent required) from both 88

hospitalised and community patients aged 70 and over. The term ‘hospitalised patient’ is used 89

to describe samples sent to the NUH clinical laboratory from inpatient departments and the 90

term ‘community patient’ is used to describe patient samples from GP’s surgeries and out 91

patient departments. All bacterial species were identified using API identification kits 92

(Biomerieux). 93

94

Multilocus Sequence Typing 95

MLST was carried out on 121 E. coli isolates using the Achtman typing scheme 96

(http://mlst.ucc.ie/mlst/dbs/Ecoli), adhering to the protocols published on the website. Briefly 97

the seven house-keeping genes, adk, fumC, gyrB, icd, mdh, recA and purA, were amplified 98

using a PCR protocol and the amplicons sequenced using the amplification primers. 99

Individual gene sequences were searched against the database and assigned an allelic 100

profile number. Sequence type (ST) designations were based upon the combination of 7 101

alleles that each strain possessed. Bionumerics v.6.5 was used to generate a minimum 102

spanning tree (MST) from non-concatenated sequences of the 7 alleles. Therefore the MST 103

illustrates clusters of closely related organisms and demonstrates the level of heterogeneity in 104

terms of allele types present and not single nucleotide polymorphisms or phylogenetic 105

relationship. 106

107

Antibiotic susceptibility testing 108

Page 4 of 18



http://mlst.ucc.ie/mlst/dbs/Ecoli


Antibiotic susceptibility profiles were obtained for all isolates using breakpoint antibiotic media. 109

The BSAC method for antimicrobial susceptibility testing was followed to prepare 110

standardised inocula.14

The antibiotic panel used was as follows; Gentamicin (2µg/ml), 111

Cefotaxime (1 µg/ml), Ceftazidime (1 µg/ml), Meropenem (2 µg/ml), Piperacillin-tazobactam 112

(16 µg/ml), Co-amoxiclav (32 µg/ml), Trimethoprim (2 µg/ml), Ciprofloxacin (4 µg/ml), 113

Cephradine (32 µg/ml), Nitrofurantoin (32 µg/ml), and Amoxicillin (32 µg/ml). All strains were 114

tested for the ESBL production phenotype using ESBL combination ID discs (MAST). All 115

isolates were also screened for the presence of the β-lactamase genes, blaTEM, blaSHV, blaCTX-116

M and blaOXA using a previously described multiplex PCR.15

Reference strains E. coli NCTC 117

13351, E. coli NCTC 13353 and Klebsiella pneumoniae NCTC 13368 were included as 118

controls. For the typing of CTX-M genes PCR products were amplified using a previously 119

published protocol,16

and amplicons were sequenced using the amplification primers. 120

121

Detection of virulence associated genes 122

The presence of 30 virulence associated genes (VAGs) was determined using previously 123

published multiplex PCR protocols,17

which include genes involved in iron acquisition, 124

adhesion, toxins and pathogenicity island markers among others. 125

126

Cell cultures 127

T24 human epithelial cells (HPA cultures) were grown in McCoy’s 5A modified media (Sigma, 128

UK) supplemented with 10% fetal bovine serum (Sigma, UK) and 0.75% L-glutamine (Sigma, 129

UK). Cells were grown in a 5% CO2 atmosphere at 37oC and sub-cultured twice-weekly. Two 130

days prior to cell infection assays, the T24 cells were seeded into 24-well plates 131

132

Association and invasion assays 133

All strains were assayed for their ability to invade human bladder epithelial cells using a 134

classical gentamicin protection assay as described previously.13

Assays were performed in 135

triplicate wells in each assay and in duplicate on different days. Bacteria were cultured 136

overnight in LB broth, harvested by centrifugation and re-suspended in supplemented tissue 137

culture medium. Bacterial cell density was then adjusted to 2x107 cfu/ml, giving an MOI of 138

Page 5 of 18




1:100. The invasive ExPEC reference strain, CFT073 was used as a positive control strain in 139

all assays and E.coli DH5α was used as a negative control strain. The mean number of 140

invasive bacteria was determined by Miles & Misra plate counts from triplicate wells. Strains 141

that showed more than a 10-fold increase in invasion compared to CFT073 were classed as 142

highly-invasive strains. Those that showed more than a 10-fold decrease in invasion 143

compared to CFT073 were deemed to be strains of limited invasive potential. 144

145

Statistical analysis 146

Statistical analysis and production of heat maps was performed using SPSS PASW statistics 147

software (version 18.0). χ 2 tests were performed to compare bacterial prevalence in different 148

patient groups and the distribution of antimicrobial resistance and VAGs among strain types. 149

150

Results 151

UTI of the elderly is caused by an epidemiologically diverse population of E. coli 152

Of the 250 urine culture plates collected from NUH, 158 originated from the community and 153

92 were from hospitalised patients. E. coli was present in 60% of sample cultures compared 154

to Enterococcus faecalis (45%), Proteus mirabilis (23%), Pseudomonas aeruginosa (20%), 155

and Staphylococcus aureus (18%). There was a significant difference between the 156

prevalence of E. coli in male (50/117) and female (100/133) cultures (P<0.001), but no 157

significant difference in the prevalence of E. coli in samples from hospitalised patients (55%) 158

or the community (63%). Sequence types (STs) were obtained for 121 E. coli isolates and 159

uploaded to the E. coli MLST database (http://mlst.ucc.ie/mlst/dbs/Ecoli). A total of 52 STs 160

were identified within the strain collection, including 11 novel STs. The most highly 161

represented STs within the strain collection were the ST131 complex, which includes ST131, 162

ST1461 and the novel ST1982, and accounted for 22% of the isolates. ST73 and ST69 163

accounted for 11% and 9% of isolates respectively. A predominant ExPEC sequence type 164

complex present in the MLST database, the ST95 complex was underrepresented in this 165

strain set (3% of isolates). The ST131 complex showed a strong association with the 166

community with 78% of isolates originating from patients within the community, compared 167

Page 6 of 18




with 22% of samples from hospitalized patients (figure 1); this association however was not 168

significantly different to observed distributions in other sequence types. 169

170

E. coli from hospital and community environments do not exhibit significantly different 171

levels of antibiotic resistance or VAG carriage but do show an association with CTX-M 172

carriage 173

The highest levels of resistance were observed against ampicillin (45% of strains), 174

trimethoprim (41%), cephradine (26%) and ciprofloxacin (21%). Slight differences were 175

observed between the resistance levels of hospital and community isolates although these 176

differences were not statistically significant. With regards to β-lactamase gene carriage 6% of 177

the E. coli isolates harboured blaSHV, 52% blaTEM (although it is not known which proportion of 178

these fall into the extended spectrum category), 11% possessed blaCTX-M, and 7% blaOXA. 179

Community strains possessed CTX-M significantly more frequently than their counterparts of 180

hospital origin (P=0.008). The most frequently detected VAGs in the strain set (figure 2b) 181

were the adhesin fimH (113/121), iron acquisition genes fyuA and iutA (98/121 and 64/121), 182

the pathogenicity island marker PAI (85/121), the serum resistance gene traT (70/121), and 183

the type II capsule marker kpsMTII (65/121). Possession of sub units of the pap operon, papA, 184

papC and papG, which are associated with adhesion to the upper urinary tract, was markedly 185

lower than fimH possession, which is involved in adhesion to the lower urinary tract (30-40% 186

of isolates carried pap genes compared to 94% fimH). This difference was observed across 187

the strain set and no significant difference was observed in the prevalence of any gene 188

between community and hospital derived strains. 189

190

There is evidence of a correlation between clonal group and antibiotic resistance 191

The distribution of resistance to trimethoprim, ciprofloxacin, ampicillin and cephradine was not 192

evenly distributed across different clonal groups (Kruskal Wallis test for independent samples, 193

P<0.05). More specifically the largest percentage of resistant strains (classed as strains with 194

the widest range of resistances to the 11 antibiotics on the panel) belonged to ST131 (Figure 195

3a). Within the ST131 complex 74% of isolates were classed as resistant to trimethoprim and 196

ciprofloxacin and 70% were resistant to ampicillin. Thirty-seven percent of isolates within the 197

Page 7 of 18




ST131 complex were resistant to over five antibiotics on the panel. When compared to non-198

ST131 isolates, the ST131 complex strains are significantly more resistant to cefotaxime 199

(P=0.012), ampicillin (P=0.004), trimethoprim (P<0.001) and ciprofloxacin (P<0.001). 200

Interestingly the ST131 complex also had a high carriage rate of β-lactamase genes, 201

accounting for 25% of blaTEM+ strains, 46% of blaCTX-M

+ strains and 63% of blaOXA

+ strains. 202

When compared to non-ST131 strains, the ST131 complex possesses significantly more 203

blaCTX-M (P=0.009) and blaOXA (P=0.014). Overall 26% of ST131 strains possessed blaCTX-M, 204

which was the highest prevalence of any of the sequence types from this study. For this 205

reason all blaCTX-M genes present in ST131 strains were typed and were all subsequently 206

confirmed to be CTX-M-15. Interestingly the only strains that demonstrated phenotypic ESBL 207

activity were the E. coli ST131 that possessed CTX-M-15. Other blaCTX-M+ strains from 208

different clonal groups demonstrated no phenotypic extended spectrum resistance. 209

210

There is no correlation between invasive potential and virulence gene carriage 211

Although some genes such as fimH, iutA, fyuA, kpsMTII II, traT and the pathogenicity island 212

were highly prevalent across all sequence types, the amount of VAGs possessed varied by 213

sequence type. ST’s 12, 1262 and 127, which exhibit low levels of antibiotic resistance, 214

possess the highest levels of VAGs (11, 10 and 9 VAGs on average respectively). The highly 215

antibiotic resistant ST131 in comparison possessed on average only 6 VAGs, and these were 216

all VAGs common to the other sequence types. The ST131 complex does not appear to 217

possess a specific gene set to which its highly virulent nature can be attributed. To determine 218

whether the presence of a specific set of virulence factors had an impact on the invasive and 219

therefore pathogenic nature of the bacterium the invasive potential of the strains was 220

determined using gentamicin protection assays. Bacterial strains were designated as highly 221

invasive if they demonstrated levels of invasion over 10-fold higher than that of the reference 222

strain, CFT073, which exhibited variation of less than 1 log across all assays performed (less 223

than 10 fold). Strains that demonstrated invasion levels 10-fold lower than CFT073 were 224

deemed those of limited invasive potential. In total 24 strains (20%) exhibited a low invasive 225

phenotype, 48 (40%) invaded within a 1-log range of CFT073 and 49 (40%) were designated 226

as highly invasive. A highly invasive phenotype could not be attributed to a specific set of 227

Page 8 of 18




VAGs. However, sfa/focDE, papC and papG allele III were significantly more prevalent in 228

strains with limited invasive potential than those with a highly invasive phenotype (P<0.001, 229

P=0.004 and P=0.01 respectively, Χ2 with 95% confidence). 230

Within the ST131 complex 56% of strains exhibited a highly invasive phenotype, 30% 231

demonstrated an invasive capability within a 1-log range of that of CFT073 and 14% 232

demonstrated limited invasive potential. When compared to the rest of the population, strains 233

within the ST131 complex did not invade to significantly higher numbers (Student’s t-test, 234

95% confidence P=0.122). 235

236

Discussion 237

This investigation set out to characterise a population of E. coli causing UTI in hospitalised 238

and community elderly patients within the Nottingham area. It was found that the population 239

was extremely diverse epidemiologically and consisted of 52 different sequence types. The 240

most frequently encountered clonal group was the ST131 complex which has links to the 241

community and ESBL production. It has been well reported that a reservoir of ESBL-242

producing organisms, which usually cause urinary tract infections, exists in the community in 243

long-term care facilities.7,12,18

Whereas no significant difference was observed in the 244

antimicrobial resistance patterns of the two groups, there was an association with CTX-M 245

gene carriage and the community location. This can be linked to the high representation of 246

ST131 within the dataset as E. coli ST131 is known to be responsible for the world-wide 247

dissemination of CTX-M-15.10,11,19

Within the ST131 complex significantly higher levels of 248

resistance to the front line antibiotics ciprofloxacin, trimethoprim, ampicillin and cefotaxime 249

was observed when compared to non-ST131 strains. The ST131 complex also had a 250

significant association with CTX-M and OXA gene carriage, thus explaining the significant 251

association with CTX-M and the community. It is not surprising that all blaCTX-M genes detected 252

in ST131 were CTX-M-15, but it is somewhat surprising however that a higher rate of carriage 253

was not observed.7,20

It is reported that CTX-M-15 is only associated with certain ST131 254

clusters, differentiated by pulse type,21

and this is the focus of further investigation to 255

determine if a specific cluster is over-represented in this population. 256

257

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With regards to virulence associated gene possession it was found that fimH, iutA, fyuA, 258

kpsMTII, traT and PAI were present in high levels across the whole population, and no 259

individual VAG profiles could be perceived for any of the sequence types. The sequence 260

types that possessed the most VAGs on average were those that possess low levels of 261

antibiotic resistance, ST12, ST127 and ST1262. It has been suggested that ST131 goes 262

against the age old ethos that a pathogen can be either antibiotic resistant or possess high 263

levels of virulence due to the enormous cost of maintaining both traits,22,23

with previous 264

studies linking ST131 with increased level of VAG carriage compared to ExPEC of other ST 265

complexes.24

In this study however the ST131 complex, which is highly antibiotic resistant, 266

does not seem to possess any specific set of VAGs nor does it possess those VAGs common 267

to other UPECs in significantly higher levels than the other sequence types. This would 268

suggest that the key to ST131’s elevated virulence potential cannot be generalised to a 269

specific VAG set or increased carriage of VAGs in general. The general hypothesis that 270

ST131 strains exhibit increased virulence which cannot be attributed to a specific gene set 271

was tested by comparing the VAG profiles of a group of strains with high, normal and limited 272

invasive potential. Strains within the ST131 complex did not exhibit an invasive potential at 273

significantly higher levels than other ExPEC strains from different ST complexes. This not 274

only suggests that the high levels of virulence reported in ST131 are not a result of invasive 275

potential but also cannot be attributed to the possession of any unique VAGs or the 276

possession of any VAGs to any greater extent than that of other ExPEC strains. It is clear that 277

an in depth study of the specific genomic content of the ST131 population is required, and is 278

the focus of ongoing work in our laboratory. 279

280

This investigation found that vast levels of heterogeneity can be observed within a single 281

population of E. coli causing UTI of the elderly. Any association between patient location and 282

antibiotic resistance was attributed to the prominent presence of the ST131 complex within 283

the dataset, which exhibited significantly higher levels of antibiotic resistance and ESBL gene 284

carriage than any other sequence type. No individual virulence associated gene profiles 285

specific to sequence types were observed and the ST131 complex does not possess any 286

VAG at higher levels than other sequence types, nor could VAG possession or invasive 287

Page 10 of 18




potential be seen to be responsible for the reported high levels of virulence in the ST131 288

complex. In short E. coli ST131 continues to emerge as an increasing threat to human health 289

due to its increasing prevalence and high rates of antimicrobial resistance, but there is no 290

clear pattern of virulence associated gene carriage which marks the ability of this ST to 291

predominate and exhibit increased virulence. 292

293

Acknowledgements 294

We would like to thank Prof David Gally, University of Edinburgh for kindly supplying strain 295

CFT073. We also acknowledge the work of Lynne Richardson and the sequencing facility at 296

University of Oxford zoology department for performing MLST sequencing reactions. 297

298

Funding 299

This work was supported by a Dowager Countess Eleanor Peel Trust grant awarded to A. M. 300

and grant 05/FE1/B882 from the Science Foundation Ireland to M. A. 301

302

Transparency declaration 303

The authors declare no competing or financial interests in this work. 304

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Single Locus Variant

Double Locus Variant

Triple Locus Variant

Quadruple Locus Variant

Branches

More than Quadruple

Locus Variant

Nodes

Community patients

Hospitalised patients

ST12 Sequence type

ST131

ST127

ST38ST69

ST12

ST73

ST1262

ST14

Novel sequence types from

this studyST1975

ST1982ST999

ST429

ST1461

ST538

ST80

ST1976

ST1981

ST68

ST405

ST1567

ST393

ST1974

ST1978

ST1979

ST59

ST95ST372ST1523

ST421 ST140

ST1983

ST929 ST144

ST420

ST1985

ST141

ST550 ST1975

ST404

ST1304

ST58

ST962

ST1297

ST1972ST295

ST607

ST205

ST1973

ST93ST648

ST10

ST48

ST297

Single Locus Variant

Double Locus Variant

Triple Locus Variant

Quadruple Locus Variant

Branches

More than Quadruple

Locus Variant

Nodes

Community patients

Hospitalised patients

ST12 Sequence type

ST131

ST127

ST38ST69

ST12

ST73

ST1262

ST14

Novel sequence types from

this studyST1975

ST1982ST999

ST429

ST1461

ST538

ST80

ST1976

ST1981

ST68

ST405

ST1567

ST393

ST1974

ST1978

ST1979

ST59

ST95ST372ST1523

ST421 ST140

ST1983

ST929 ST144

ST420

ST1985

ST141

ST550 ST1975

ST404

ST1304

ST58

ST962

ST1297

ST1972ST295

ST607

ST205

ST1973

ST93ST648

ST10

ST48

ST297

372

Fig. 1. Minimum spanning tree of E. coli sequence types from elderly urinary tract infections. 373

Grey shading indicates proportion of isolates within ST derived from hospital environments 374

and un-shaded areas indicate proportion of isolates within ST derived from community 375

environments. Strains within the same ST possess 7 identical alleles, whereas a single locus 376

variant possesses one different allele to the other ST and a double locus variant differs by two 377

alleles. 378

379

Page 15 of 18




0.010.020.030.040.050.060.070.080.090.0

100.0

pap

A

pap

C

pap

EF

pap

G I

pap

G I

I,

pap

G

pap

G

pap

G

sfa/f

ocD

E

sfaS

focG

afa

/dra

BC

bm

aE

gafD

nfa

E

fim

H

hly

A

cn

f1

cd

tB

fyu

A

iutA

kp

sMT

II

kp

sMT

K1

k5

rfc

ibeA

cv

aC

traT

PA

I

Gene

Perc

en

tag

e p

rev

ale

nce

0

10

20

30

40

50

60

bla

TE

M

AM

P

TR

M

CE

P

CIP

NT

CT

X

CA

Z

bla

CT

X-M GM

PIP

-TA

Z

bla

SH

V

bla

OX

A

AU

G

ME

R

Antibiotic

Perc

enta

ge

of

resi

stan

t st

rain

s

(a)

(b)

0.010.020.030.040.050.060.070.080.090.0

100.0

pap

A

pap

C

pap

EF

pap

G I

pap

G I

I,

pap

G

pap

G

pap

G

sfa/f

ocD

E

sfaS

focG

afa

/dra

BC

bm

aE

gafD

nfa

E

fim

H

hly

A

cn

f1

cd

tB

fyu

A

iutA

kp

sMT

II

kp

sMT

K1

k5

rfc

ibeA

cv

aC

traT

PA

I

Gene

Perc

en

tag

e p

rev

ale

nce

0

10

20

30

40

50

60

bla

TE

M

AM

P

TR

M

CE

P

CIP

NT

CT

X

CA

Z

bla

CT

X-M GM

PIP

-TA

Z

bla

SH

V

bla

OX

A

AU

G

ME

R

Antibiotic

Perc

enta

ge

of

resi

stan

t st

rain

s

(a)

(b)

380

Fig. 2. (a) Antibiotic resistance prevalence in urinary E. coli isolates of hospital and 381

community origin. Black bars, hospital isolates; white bars, community isolates. (b) Virulence 382

associated gene prevalence in urinary E. coli isolates of hospital and community origin. Black 383

bars, hospital isolates; white bars, community isolates. 384

Page 16 of 18




papG

I

38

69

131

Others

73

14

127

1262

12

fimH

fyuA

PA

I

traT

kpsM

TII

iutA

pap

A

pap

C

sfa/fo

cD

E

pap

G allele III

K5

pap

G II &

III

hly

A

focG

pap

G allele II

pap

EF

cnf1

ibeA

rfc

K1

sfaS

kpsM

TIII

cvaC

pap

G allele I

afa

/dra

BC

bm

aE

gafD

nfa

E

cdtB

Virulence Associated Gene

Seq

uen

ce T

yp

e

Percentage

prevalence

100 %

40 %

20 %

0 %

80 %

60 %

pap

G I

100 %

20 %

0 %

80 %

60 %

40 %

38

69

131

Others

73

14

127

1262

12

Seq

uen

ce T

yp

e

TR

M

bla

TE

M

AM

P

CE

P

CIP

CA

Z

CT

X

NT

GM

bla

SH

V

PIP

/TA

Z

bla

CT

X-M

bla

OX

A

AU

G

ME

R

Antibiotic/ antibiotic resistance gene(b)

(a)

papG

I

38

69

131

Others

73

14

127

1262

12

fimH

fyuA

PA

I

traT

kpsM

TII

iutA

pap

A

pap

C

sfa/fo

cD

E

pap

G allele III

K5

pap

G II &

III

hly

A

focG

pap

G allele II

pap

EF

cnf1

ibeA

rfc

K1

sfaS

kpsM

TIII

cvaC

pap

G allele I

afa

/dra

BC

bm

aE

gafD

nfa

E

cdtB


Seq

uen

ce T

yp

e

Percentage

prevalence

100 %

40 %

20 %

0 %

80 %

60 %

pap

G I

papG

I

38

69

131

Others

73

14

127

1262

12

fimH

fyuA

PA

I

traT

kpsM

TII

iutA

pap

A

pap

C

sfa/fo

cD

E

pap

G allele III

K5

pap

G II &

III

hly

A

focG

pap

G allele II

pap

EF

cnf1

ibeA

rfc

K1

sfaS

kpsM

TIII

cvaC

pap

G allele I

afa

/dra

BC

bm

aE

gafD

nfa

E

cdtB


Seq

uen

ce T

yp

e

Percentage

prevalence

100 %

40 %

20 %

0 %

80 %

60 %

pap

G I

100 %

20 %

0 %

80 %

60 %

40 %

38

69

131

Others

73

14

127

1262

12

Seq

uen

ce T

yp

e

TR

M

bla

TE

M

AM

P

CE

P

CIP

CA

Z

CT

X

NT

GM

bla

SH

V

PIP

/TA

Z

bla

CT

X-M

bla

OX

A

AU

G

ME

R

Antibiotic/ antibiotic resistance gene

100 %

20 %

0 %

80 %

60 %

40 %

38

69

131

Others

73

14

127

1262

12

Seq

uen

ce T

yp

e

TR

M

bla

TE

M

AM

P

CE

P

CIP

CA

Z

CT

X

NT

GM

bla

SH

V

PIP

/TA

Z

bla

CT

X-M

bla

OX

A

AU

G

ME

R

Antibiotic/ antibiotic resistance gene(b)

(a)

385

Fig. 3. (a) Heat map of antimicrobial resistance distribution across predominant E. coli 386

sequence types. Darker shaded areas indicate higher prevalence. Abbreviations; blaTEM, TEM 387

β-lactamase gene; blaSHV, SHV β-lactamase gene; blaCTX-M, CTX-M β-lactamase gene; blaOXA, 388

OXA β-lactamase gene; GM, Gentamicin; CTX, Cefotaxime; CAZ, Ceftazidime; MER, 389

Meropenem; PIP-TAZ, Piperacillin-tazobactam; AUG, Augmentin; TRM, Trimethoprim; CIP, 390

Ciprofloxacin; CEP, Cephradine; NT, Nitrofurantoin; AMP, Ampicillin. (b) Heat map of 391

virulence associated gene distribution across predominant E. coli sequence types. 392

Page 17 of 18




0.010.020.030.040.050.060.070.080.090.0

100.0

pap

A

pap

C

pap

EF

pap

G I

pap

G I

I, I

II

pap

G a

llel

e I

pap

G a

llel

e II

pap

G a

llel

e II

I

sfa/

focD

E

sfaS

focG

afa/

dra

BC

bm

aE

gaf

D

nfa

E

fim

H

hly

cnf1

cdtB

fyu

A

iutA

kpsM

T I

I

kpsM

T I

II K1

k5

rfc

ibeA

cvaC

traT

PA

I


Perc

en

tag

e p

rev

ale

nce

393

Fig. 4. Virulence associated gene prevalence in E. coli isolates with different invasive 394

potentials. White bars, isolates with low invasive potential; hatched bars, strains with normal 395

invasive potential comparable to that of CFT073; black bars, strains with high invasive 396

potential. 397

398

Page 18 of 18



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Journal of Antimicrobial Chemotherapyirep.ntu.ac.uk/id/eprint/6481/1/205594_7750 Mcnally Postprint.pdf · Journal of Antimicrobial Chemotherapy: under review Journal of Antimicrobial