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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
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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: alan.mcnally@ntu.ac.uk 21
22
23
24
25
26
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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
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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
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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
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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
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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
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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
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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
Journal of Antimicrobial Chemotherapy: under review
Journal of Antimicrobial Chemotherapy
Confidential: for peer review only
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
Journal of Antimicrobial Chemotherapy: under review
Journal of Antimicrobial Chemotherapy
Confidential: for peer review only
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
Virulence Associated Gene
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
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
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
Journal of Antimicrobial Chemotherapy: under review
Journal of Antimicrobial Chemotherapy
Confidential: for peer review only
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
Virulence Associated Gene
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
Journal of Antimicrobial Chemotherapy: under review
Journal of Antimicrobial Chemotherapy