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1
Virulence potential of Escherichia coli isolates from skin and soft 1
tissue infections (SSTI) 2
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Running title: E. coli from SSTI 4
5
Živa PETKOVŠEK1, Kristina ELERŠIČ2, Marija GUBINA3, Darja ŽGUR-6
BERTOK1, Marjanca STARČIČ ERJAVEC1* 7
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The work was done at the Department of Biology, Biotechnical Faculty, University of 10
Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia and at the Institute of 11
Microbiology and Immunology, Medical Faculty, University of Ljubljana, Zaloška 4, 12
1000 Ljubljana, Slovenia. 13
14
1 Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 15
111, 1000 Ljubljana, Slovenia 16
2 Department of Surface Engineering, and Optoelectronics, Institut Jožef Stefan, 17
Teslova ulica 30, 1000 Ljubljana, Slovenia 18
3 Institute of Microbiology and Immunology, Medical Faculty, University of 19
Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia. 20
21
* Corresponding author. Mailing address: Department of Biology, Biotechnical 22
Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia. Phone: 23
+386 1 423 33 88. Fax: +386 1 257 33 90. E-mail: marjanca.starcic.erjavec@bf.uni-24
lj.si 25
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01421-08 JCM Accepts, published online ahead of print on 8 April 2009
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ABSTRACT 26
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Escherichia coli (E. coli) strains are frequently isolated from skin and soft tissue 28
infections (SSTI) however, their virulence potential has not yet been extensively 29
studied. In the present study we characterized 102 E. coli SSTI strains mostly isolated 30
from surgical and traumatic wounds, ulcus cruris and decubitus. The strains were 31
obtained from the Institute of Microbiology and Immunology, University of 32
Ljubljana, Slovenia. Phylogenetic background, virulence factors (VFs), and antibiotic 33
resistance profiles were determined. Correlations between VFs and phylogenetic 34
groups were established and analyzed with regard to patient factors. Further, 35
associations of the three most prevalent antibiotic resistances with virulence potential 36
were analyzed. Our results showed that the majority of the studied strains (65%) 37
belonged to the B2 phylogenetic group. The most prevalent VF was ompT (80%), 38
while toxin genes cnf1 and hlyA were found with prevalences of 32% and 30%, 39
respectively. None of the investigated bacterial characteristics were significantly 40
associated with patient gender, age, type of infection, or immunodeficiency. The most 41
prevalent was resistance to ampicillin (46%), followed by resistance to tetracycline 42
(25%) and fluoroquinolones (21%). Strains resistant to ciprofloxacin exhibited a 43
significantly reduced prevalence of cnf1 (P<0,05) and usp (P<0,01). Our study 44
revealed that E. coli isolates from SSTI exhibit a remarkable virulence potential 45
comparable to that of E. coli isolates from urinary tract infections and bacteremia. 46
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INTRODUCTION 47
48
Skin and soft tissue infections (SSTIs) are one of the most common infections in 49
patients of all age groups. Infections are mostly self-limited or may be treated with 50
antibiotics. However, moderate or severe cases may require hospitalization and 51
parenteral therapy (30). The most common causative agents are Staphylococcus 52
aureus (S. aureus) and aerobic streptococci (9, 10, 41, 43). However, several reports 53
associating the enterobacterium Escherichia coli (E. coli) with SSTI have been 54
published: E. coli was found to be the causative agent of neonatal omphalitis (7), 55
cellulitis localized to lower or upper limbs (4, 6, 49), necrotizing fasciitis (1, 25, 28), 56
surgical site infections (44), infections after burn injuries (37), and others. A study 57
monitoring SSTI over a 7-year period encompassing 3 continents (Europe, Latin 58
America and North America) showed E. coli to be an important causative agent, since 59
it was the 3rd most prevalent isolated species, preceded solely by S. aureus and 60
Pseudomonas aeruginosa. The β-hemolytic Streptococcus spp. group was only the 7th 61
ranking pathogen in North America and Europe and 10th in Latin America (30). E. 62
coli isolates from SSTI therefore merit detailed studies, especially taking into account 63
the dramatic decline in antibiotic susceptibility of pathogenic E. coli strains in recent 64
years. Despite the need for characterization of E. coli strains from SSTI, to our 65
knowledge, only a single E. coli isolate from a deep surgical wound infection has 66
been characterized (21). The aim of our study was to characterize a larger collection 67
of E. coli isolates from SSTI. Our work was focused on their virulence potential: 68
phylogenetic distribution, virulence factor profile, and prevalence of antibiotic 69
resistances. Correlations between patient and strain characteristics were studied as 70
well as correlations between virulence factor profile and antibiotic resistance. As the 71
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strains were isolated from extraintestinal sites of infections, we screened for VFs 72
typical of extraintestinal pathogenic E. coli. 73
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MATERIALS AND METHODS 75
76
Strains. 77
The studied E. coli strains were isolated between 28.8.2006 and 30.11.2006 at the 78
Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia. 79
During this time period 3186 wound samples, swabs of eyes, ears, and genital 80
infections from patients residing in different parts of Slovenia were collected. In 216 81
(7%) of the collected samples, E. coli strains were found. One hundred and two (47%) 82
of the E. coli positive samples were from SSTI; 38 (37%) from surgical wounds, 20 83
(20%) from ulcus cruris, 14 (14%) from decubitus, 9 (9%) from traumatic wounds, 5 84
(5%) from localized skin infections in the perigenital/perianal region, 5 (5%) from 85
fistula (2 fistulae were perianal, 1 was inguinal and 2 were from skin at the site of 86
radiotherapy), 4 (4%) from omphalitis, 4 (4%) from diabetic wounds, 1 (1%) from a 87
burn infection, 1 (1%) from a radiation wound, and 1 (1%) from a pustula. Thirteen 88
(13%) E. coli strains were from monomicrobial SSTI and 89 (87%) E. coli strains 89
were from polymicrobial SSTI. Fifty eight (57%) of the isolates were obtained from 90
male and 44 (43%) from female patients. The mean age of the patients was 51.1 years; 91
20 (20%) of the patients were children younger than 19 years, 14 (14%) were between 92
19-45, 26 (25%) were between 46-65, and 42 (41%) patients were older than 65 years. 93
In 63 (62%) cases, the infection was acute and in the remaining 39 (38%) chronic. 94
The infection was regarded as chronic, if (i) the wound remained unhealed for at least 95
3 months or if the diagnosis was either (ii) ulcus cruris, (iii) decubitus or (iv) diabetic 96
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gangrene. Patients were regarded as immunocompromised if they: (i) suffered from a 97
chronical wound and were older than 65 years, (ii) were neonates (aged up to 4 98
weeks), (iii) suffered from diabetis and a wound(s), (iv) were undergoing treatment 99
for cancer at the Institute of Oncology, Ljubljana, (v) were undergoing treatment at 100
the Institute for rehabilitation, Ljubljana and suffered from decubitus for more than 6 101
months. Fifty three (52%) of the patients were immunocompromised. A single strain 102
from each patient was analyzed. 103
104
Phylogenetic analysis. 105
The isolates were assigned to one of the four main phylogenetic groups (A, B1, B2 or 106
D) by multiplex PCR, as described by Clermont et al. (5). 107
108
Virulence factor profiling. 109
All isolates were tested for the following VFs: cnf1 (cytotoxic necrotizing factor 1), 110
hlyA (hemolysin), papA, papGII and papGIII (P-fimbriae), sfaDE (S-fimbriae), 111
afa/dra (Afa/Dr adhesins), iucD (aerobactin), kpsMT (group II capsule), ompT (outer 112
membrane protease), and usp (uropathogenic specific protein). Prior to genotyping, 113
boiled lysates were prepared (27). The PCR mix (25 µL) contained template DNA (5 114
µL), primers at a concentration of 0,8 µM, dNTP (0,2 mM), MgCl2 (2,5 mM) and Taq 115
DNA polymerase (5U/µL) in 1× Taq DNA buffer. The primers and PCR programs 116
used were described previously (14, 19, 22, 23, 26, 27, 32, 48). Dot – blot 117
hybridization was performed to confirm the PCR results. Positive and negative 118
controls were included. To calculate the VF score, alleles papA, papGII and papGIII 119
were considered as a single, pap VF. Thus, if a strain was positive for at least one of 120
the studied pap alleles, it was regarded as pap positive. 121
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Antimicrobial susceptibility testing. 123
Antimicrobial sensitivity testing to the following antimicrobial agents was performed 124
by estimation of MIC values using Etest® (AB BIODISK) and the National 125
Committee for Clinical Laboratory Standards (NCCLS) interpretive criteria (33): 126
ampicillin, piperacillin, amoxicillin + clavulanic acid, piperacillin + tazobactam, 127
cefazolin, cefuroxime axetil, cefoxitin, cefixime, cefotaxime, ceftazidime, cefepime, 128
imipenem, aztreonam, gentamicin, amikacin, netilmicin, tetracycline, norfloxacin, 129
ciprofloxacin, trimethoprim + sulfamethoxazole, and ertapenem. 130
131
Statistical analysis. 132
Fisher’s exact test (two-tailed) (http://www.langsrud.com/fisher.htm) and the 133
Bonferroni correction were used to analyze the data. The threshold for statistical 134
significance after Bonferroni correction was set at P values of < 0,05. 135
136
RESULTS 137
138
Phylogenetic groups. 139
Our analysis showed that the majority of the studied isolates, namely 66 strains 140
(65%), belonged to group B2. Fourteen isolates (14%) were assigned to group D, 12 141
(12%) to group A, while 10 isolates (10%) belonged to group B1. 142
143
Virulence factors. 144
The most prevalent VF among our isolates was ompT, which was found in 82 (80%) 145
of the tested strains, followed by kpsMT, detected in 66 (65%) strains (Table 1). Other 146
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VFs were found in less than 50% of the isolates. The toxin genes, cnf1 and hlyA, were 147
found in approximately one third of the strains – cnf1 in 33 (32%) and hlyA in 31 148
(30%) strains. Twenty-seven (26%) of all strains, 82% of cnf1 encoding strains and 149
87% of hlyA encoding strains) strains encoded both toxins. The most prevalent 150
adhesin sequences among our isolates were P-fimbriae namely, 44 (43%) of the tested 151
strains encoded at least one of the amplified papA or papG alleles. Only one isolate 152
was found to encode afa/draBC of Afa/Dr-adhesins (1%). Analysis of the two P-153
fimbrial allelic variants for the binding adhesin (papGII and papGIII) showed that 154
both alleles were encoded with similar prevalence 10 strains (10%) and 15 strains 155
(15%), respectively (Table 1). Analysis of the major rod subunit papA showed that the 156
F10 allele was by far the most prevalent, as it was found in 26 isolates, representing 157
60% of the strains coding for a papA allele (data not shown). Eight strains (19% of the 158
papA coding strains) encoded F12-15. The papA allele F9 was found in 5 strains 159
(12%), alleles F12, F13 and F16 in 4 strains (9%), alleles F11 and F14 in 3 strains 160
(7%), allele F8 in 2 strains (4%) and the F7-2 allele in 1 strain (2%). The papA allelic 161
variants F7-1, F15 and F48 were not found in any of the studied strains. In one isolate 162
the papG gene was detected whereas papA analysis was negative for all tested alleles. 163
The number of VFs, detected in a single strain, varied from 0–8. The investigated 164
strains most commonly possessed 3–5 VFs. The average number of VFs (average VF 165
score) was 3.8 (data not shown). 166
167
Distribution of VFs among phylogenetic groups. 168
The distribution of VFs of the studied strains with regard to phylogenetic group B2 is 169
presented in Table 1. In general, strains belonging to the B2 phylogenetic group 170
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exhibited the highest prevalence of VFs, although only association of papA with the 171
B2 group was statistically significant (P<0,01; Table 1). 172
Subsequently, the correlation between VF score and phylogenetic group was 173
examined. The majority of strains encoding 6–8 VFs belonged to the B2 phylogenetic 174
group. Strains encoding 3–5 VFs were more equally distributed among all 175
phylogenetic groups. The presence of 0-2 of the tested VFs was uncommon among 176
the group B2 strains. 177
178
Distribution of phylogenetic groups and VFs in relation to patient 179
characteristics. 180
The distribution of virulence potential associated characteristics in relation to patient 181
gender, age (younger than 19, 19–45, 46–65, and older than 65), infection type (acute, 182
chronic) and immune system status (normal, weakened) was analyzed. When 183
comparing phylogenetic group, individual VFs, and VF score in relation to patient 184
characteristics not a single statistically significant association was found (Table 2). 185
The distribution of bacterial characteristics in relation to the clinical syndrome was 186
analyzed for isolates from surgical wound infections and no significant correlations 187
were found (data not shown). Other syndrome subgroups, as well as the number of 188
monomicrobial E. coli strains, were too small to perform a statistical analysis. 189
190
Antimicrobial resistance. 191
Resistance of the studied isolates to some of the most clinically relevant antibiotics 192
was tested. The most prevalent, found in 47 (46%) isolates, was resistance to 193
ampicillin, followed by tetracycline resistance and resistance to fluoroquinolones 194
(norfloxacin and ciprofloxacin), found in 25 (25%) and 21 (21%) isolates, 195
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respectively. Nineteen (19%) of the strains were resistant to piperacillin, 17 (17%) to 196
trimethoprim + sulfamethoxazole and 14 (14%) to amoxicillin + clavulanic acid. Nine 197
(9%) strains were resistant to cefazolin, a cephalosporin of the first generation, while 198
the prevalence of resistances to cephalosporins of the second (cefuroxime axetil and 199
cefoxitin), third (cefixime, cefotaxime, ceftazidime) and fourth generation (cefepime), 200
was 5% or lower. The least prevalent were resistances to piperacillin+tazobactam, 201
aztreonam, and gentamicin, as only one strain (1%) was found to be resistant. A single 202
strain was found to produce extended-spectrum beta-lactamase. All studied strains 203
were susceptible to imipenem, amikacin, netilmicin and ertapenem. 204
205
Association of antimicrobial resistance with virulence potential. 206
A correlation between virulence potential and antibiotic susceptibility/resistance was 207
studied for tetracycline, ciprofloxacin and ampicillin, the three most prevalent 208
antibiotic resistances among the studied isolates. We found that susceptible strains 209
more commonly (albeit not significantly) belonged to the B2 phylogenetic group and 210
that the prevalence of VFs among these strains was higher (Table 3) as the VF score 211
was higher. The difference in VF score between susceptible and resistant strains was 212
1.2 for tetracycline susceptible/resistant strains and 1.4 for ciprofloxacin 213
susceptible/resistant strains, while only 0.5 VF in the case of ampicillin 214
susceptibility/resistance. Strains resistant to ciprofloxacin significantly less frequently 215
harbored cnf1 (P<0.05) and usp (P<0.01). Other significant differences were not 216
found (Table 3). 217
Twelve (12%) of the studied isolates were resistant to both tetracycline and 218
ciprofloxacin and 67 strains (66%) were susceptible to both antibiotics. Virulence 219
profile analysis showed that the strains resistant to both tetracycline and ciprofloxacin 220
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possessed the least VFs as they on average possessed 2.3 VFs (data not shown). ompT 221
was found to be negatively associated with resistance to both tetracycline and 222
ciprofloxacine (P<0.05). In general, VFs were concentrated in strains susceptible for 223
both antibiotics as the average VF score of the susceptible strains reached a value of 224
4.2 (data not shown). Statistically significant correlations between susceptible strains 225
and possession of hlyA (P<0.05) and usp (P<0.01) sequences were found. Strains, 226
resistant to a single antibiotic, reached a VF score of 3.5 (Cipr) and 3.6 (Tcr) (data not 227
shown). 228
229
DISCUSSION 230
231
Even though E. coli is the most frequently isolated enterobacterium from SSTI, to our 232
knowledge, this is the first study of the virulence profile and antibiotic susceptibility 233
of a larger strain collection of SSTI E. coli strains. Since the studied strains were 234
isolated from extraintestinal sites of infection, we assumed that their virulence 235
potential would be similar to that of other ExPEC (extraintestinal pathogenic E. coli) 236
strains. ExPEC strains differ significantly from both intestinal pathogens and 237
commensal strains, as they possess typical virulence determinants (adhesins, e.g. P 238
fimbriae, iron-acquisition systems, e.g. aerobactin, host defense-avoidance 239
mechanisms, e.g. capsule, toxins, e.g. hemolysin) and belong mainly to phylogenetic 240
group B2 or, to a lesser extent, to group D (36, 38). 241
An assessment of our results and those of other studies on ExPEC and human fecal E. 242
coli isolates is presented in Table 4. Compared to fecal isolates, the strains 243
investigated in our study more frequently belonged to the B2 phylogenetic group and 244
exhibited a higher prevalence of the tested VFs. In addition, a virulence factor profile 245
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similar to that of other ExPEC subgroups is evident. Nonetheless, some discrepancies 246
in prevalence of individual VFs are obvious, namely, the prevalence of usp among 247
SSTI isolates was somewhat lower, 44%, while Bauer et al. (2) reported prevalences 248
of 69% for urinary tract infection (UTI) strains, 74% for periurethral isolates and 58% 249
for vaginal isolates. Previously, in an experimental mouse model, the USP protein was 250
shown to contribute to UTI (47) therefore, the observed lower prevalences among non 251
UTI isolates are not surprising. On the other hand, discrepancies might also be due to 252
geographic differences in distribution of VFs. Differences in virulence factor profiles 253
between distinct populations have previously been reported among cat populations 254
from distant locations. Among feline uropathogenic E. coli strains from the United 255
Kingdom a 41% prevalence of papGII + papGIII was determined while among feline 256
uropathogenic E. coli strains from New Zealand, the prevalence of papGII + papGIII 257
was 100% (8). 258
It is noteworthy that the prevalences of cnf1 and hlyA (32% and 30%, respectively) 259
were similar to those found among UTI isolates (Table 4) indicating, that CNF1 could 260
also play a significant role in SSTIs. While it is well established that CNF1 is a 261
urotoxin, a recent study showed that in vitro CNF1 also blocks intestinal epithelial 262
wound repair (3). cnf1 is known to be associated with hlyA in the pathogenicity island 263
PAI II J96 (40), the high prevalence of hlyA (31%) is therefore probably due to the 264
presence of PAI sequences, as more than 80% of the strains harbored both toxin genes 265
cnf1 and hlyA. Whether hemolysin, a well established urotoxin, might be important 266
for instigating SSTIs has, to our knowledge, not yet been investigated. The same can 267
be stated for all other tested VFs. 268
Further, new not yet identified, VFs could play a significant role in SSTIs. For 269
example, our study indicates that new papA alleles might be harbored by some 270
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isolates, since from a strain that encoded papG adhesins no papA alleles could be 271
amplified in the multiplex PCR designed by Johnson et al. (23). 272
It is well established that host characteristics can play a decisive role in the 273
development of disease. To ascertain whether there is an association between patient 274
characteristics and virulence potential of the studied strains we analyzed gender, age, 275
infection type and immune system status in relation to virulence potential. No 276
significant differences were observed. However, further studies on larger strain 277
collections are needed especially as in our study we could not distinguish patients that 278
are imunocompromised due to treatment with steroids and neutropenics. 279
Resistance of pathogens against antimicrobial agents is a global health care problem 280
and a subject of intense research. A number of studies have reported a significantly 281
reduced virulence potential among UPEC isolates resistant to certain antibiotics such 282
as quinolones, chloramphenicol, tetracycline and others, (12, 15, 17, 29, 31, 42, 45, 283
46), but not among ampicillin, and trimethoprim resistant isolates (16, 46). In our 284
study, the most prevalent were resistances to ampicillin, tetracycline, norfloxacin, 285
ciprofloxacin, piperacillin, trimethoprim + sulfamethoxazole and amoxicillin + 286
clavulanic acid. These antibiotics were, or still are, of high clinical significance and 287
therefore, higher resistance prevalences are not surprising. The virulence potential in 288
relation to antibiotic susceptibility/resistance was analyzed for tetracycline, 289
ciprofloxacin and ampicillin, as resistances to these three antibiotics were the most 290
prevalent. The results of our analysis are in agreement with previous studies 291
performed on urinary tract infection isolates (13, 31, 35, 42), demonstrating a lower 292
virulence potential among strains resistant to tetracycline and ciprofloxacin, while 293
ampicillin resistant strains exhibited no loss in virulence potential. Several hypothesis 294
have been postulated to explain such differences in VF profile (15, 29, 35, 46) 295
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however, it is currently believed that the differences observed between VF profiles of 296
antibiotic susceptible and resistant strains are due to differences in population source. 297
It has been suggested that ecological factors determine associations and that antibiotic 298
resistant strains may derive from an animal or environmental source rather than from 299
human fecal E. coli (15, 16). An exception are ampicillin resistant isolates which may 300
derive from susceptible strains via acquisition of transferable resistance elements, 301
with no major changes in VF profile (16). 302
In conclusion, the studied E. coli strains from SSTIs exhibited a remarkable virulence 303
potential indicating their medical significance. Even though additional, in vivo studies, 304
should be performed to confirm the significance of the detected VFs, we believe that 305
E. coli strains should be considered as important causative agents of SSTI. Further 306
analysis of VF profiles with regard to specific clinical syndromes and defined severity 307
is recommended. 308
309
310
ACKNOWLEDGEMENTS 311
We are grateful to Olga Križaj for providing the investigated strains and to prof. dr. 312
Andrej Blejec for his help with statistical analysis. This research was financed by 313
Grant P1-0198 from the Slovenian Research Agency (ARRS). Živa Petkovšek is a 314
recipient of a Ph.D. grant from ARRS. 315
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487
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TABLE 1. VF prevalences among phylogenetic groups. 489
Prevalence of trait (no. [%] of isolates) Phylogenetic group (no. [%] of all isolates) Virulence factor (VF) All isolates
(102 [100]) A
(12 [12]) B1 (10 [10])
B2 (66 [65])
D (14 [14])
Toxins cnf1 33 (32) 4 (33) 2 (20) 22 (33) 5 (36) hlyA 31 (30) 3 (25) 2 (20) 21 (32) 5 (36)
Fimbriae and/or adhesins papA 43 (41) 3 (25) 1 (10) 36 (55)** 3 (21) papGII 10 (10) 0 0 9 (14) 1 (7) papGIII 15 (15) 2 (17) 0 11 (17) 2 (14) sfaDE 37 (36) 2 (17) 4 (40) 25 (38) 6 (43) afa/draBC 1 (1) 0 0 1 (2) 0
Iron uptake iucD 48 (47) 7 (58) 3 (30) 34 (52) 4 (29)
Capsule kpsMT 66 (65) 6 (50) 4 (40) 48 (73) 8 (57)
Other ompT 82 (80) 7 (58) 6 (60) 58 (88) 11 (79) usp 45 (44) 4 (33) 3 (30) 35 (53) 3 (21)
Number of VFs 0 7 (7) 1 (8) 3 (30) 2 (3) 1 (7) 1 10 (10) 4 (33) 1 (10) 2 (3) 3 (21) 2 11 (11) 0 1 (10) 8 (12) 2 (14) 3 13 (13) 1 (8) 2 (20) 10 (15) 0 4 23 (23) 3 (25) 0 16 (24) 4 (29) 5 18 (18) 2 (17) 2 (20) 12 (18) 2 (14) 6 10 (10) 0 1 (10) 7 (11) 2 (14) 7 6 (6) 1 (8) 0 5 (8) 0 8 4 (4) 0 0 4 (6) 0
A correlation between a virulence factor (VF) and the phylogenetic group B2 was ascertained by comparing the prevalences of VFs between B2 and all non-B2 strains. 490
Fisher’s exact test and the Bonferroni correction were used to analyze the data. P value following Bonferroni correction is indicated by two asterisk where P is <0.01. 491
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TABLE 2. Distribution of phylogenetic groups, VFs and number of VFs in relation to patient gender, age, infection type and immune system status. 492
Prevalence (no. [%] of the tested strains) Sex
(no. [%]) Age
(no. [%]) Infection type
(no. [%]) Immune system status
(no. [%]) M F <19 19-45 46-65 65< Acute Chronic Normal Weakened
58 (57) 44 (43) 19 (19) 15 (15) 26 (25) 42 (41) 63 (62) 39 (38) 49 (48) 53 (52) Phylogenetic group A 5 (9) 7 (16) 2 (11) 3 (20) 4 (15) 3 (7) 9 (14) 3 (8) 7 (14) 5 (9) B1 5 (9) 5 (11) 2 (11) 2 (13) 3 (12) 3 (7) 5 (8) 5 (13) 3 (6) 7 (13) B2 41 (71) 25 (57) 13 (68) 7 (47) 16 (62) 30 (71) 41 (65) 25 (64) 31 (63) 35 (66) D 7 (12) 7 (16) 2 (11) 3 (20) 3 (12) 6 (14) 8 (13) 6 (15) 8 (16) 6 (11) Virulence factor
(VF)
cnf1 16 (28) 17 (39) 8 (42) 6 (40) 7 (27) 12 (29) 25 (40) 8 (21) 13 (27) 20 (38) hly 17 (29) 14 (32) 6 (32) 4 (27) 8 (31) 13 (31) 23 (37) 8 (21) 13 (27) 18 (34) papA 21 (36) 22 (50) 5 (26) 4 (27) 12 (46) 22 (52) 23 (37) 20 (51) 21 (43) 22 (42) papGII 5 (9) 5 (11) 1 (5) 0 5 (19) 4 (10) 7 (11) 3 (8) 2 (4) 8 (15) papGIII 6 (10) 9 (20) 1 (5) 4 (27) 4 (15) 6 (14) 11 (17) 4 (10) 10 (20) 5 (9) sfaDE 22 (38) 15 (34) 9 (47) 7 (47) 10 (38) 11 (26) 25 (40) 12 (31) 17 (35) 20 (38) afa/dra 1 (2) 0 1 (5) 0 0 0 1 (1) 0 1 (2) 0 iucD 29 (50) 19 (43) 8 (42) 8 (53) 11 (42) 21 (50) 29 (46) 19 (49) 21 (43) 27 (51) kpsMT 37 (64) 29 (66) 14 (74) 11 (73) 12 (46) 29 (69) 41 (65) 25 (64) 25 (51) 41 (77) ompT 45 (78) 37 (84) 15 (79) 14 (93) 17 (65) 36 (86) 49 (78) 33 (85) 40 (82) 42 (79) usp 23 (40) 22 (50) 10 (53) 9 (60) 9 (35) 17 (40) 29 (46) 16 (41) 20 (41) 25 (47) Number of VFs
0 4 (7) 3 (7) 1 (5) 0 4 (15) 2 (5) 4 (6) 3 (8) 4 (8) 3 (6) 1 7 (12) 1 (2) 2 (11) 1 (7) 4 (15) 3 (7) 5 (8) 5 (13) 6 (12) 4 (8) 2 7 (12) 4 (9) 2 (11) 2 (13) 2 (8) 5 (12) 8 (13) 3 (8) 8 (16) 3 (6) 3 7 (12) 5 (11) 1 (5) 3 (20) 2 (8) 7 (17) 7 (11) 6 (15) 5 (10) 8 (15) 4 13 (22) 10 (23) 5 (26) 3 (20) 8 (31) 7 (17) 15 (24) 8 (21) 9 (18) 14 (26) 5 10 (17) 8 (18) 3 (16) 2 (13) 1 (4) 12 (29) 9 (14) 9 (23) 8 (16) 10 (19) 6 5 (9) 5 (11) 3 (16) 1 (7) 2 (8) 4 (10) 9 (14) 1 (3) 6 (12) 4 (8) 7 2 (3) 4 (9) 1 (5) 3 (20) 1 (4) 1 (2) 3 (5) 3 (8) 2 (4) 4 (8) 8 3 (5) 1 (2) 1 (5) 0 2 (8) 1 (2) 3 (5) 1 (3) 1 (2) 3 (6)
A correlation between bacterial and patient characteristics was ascertained using Fisher’s exact test and the Bonferroni correction. No statistically significant 493
correlations were found. 494
495
496
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TABLE 3. Distribution of phylogenetic groups, VFs, and numbers of VFs in relation to resistance phenotypes. 497
498
P values following Bonferroni correction are indicated by asterisk where P is <0.05. Symbols: *, P<0.05; **, P<0.01. Two strains were not tested for 499
tetracycline resistance and one strain was not tested for ciprofloxacin resistance. These strains were therefore omitted from susceptibility/resistance analysis 500
for the two antibiotics. 501
502
Prevalence (no. [%] of strains) Tetracycline (no. [%]) Ciprofloxacin (no. [%]) Ampicillin (no. [%]) Susceptible
(75 [74]) Resistant (25 [25])
Susceptible (80 [78])
Resistant (21 [21])
Susceptible (55 [54])
Resistant (47 [46])
Phylogenetic group A 7 (9) 5 (20) 9 (11) 3 (14) 6 (11) 6 (13) B1 7 (9) 3 (12) 7 (9) 3 (14) 4 (7) 6 (13) B2 52 (69) 12 (48) 52 (65) 13 (62) 40 (73) 26 (55)
D 9 (12) 5 (20) 12 (15) 2 (10) 5 (9) 9 (19) Virulence factor (VF) cnf1 26 (35) 7 (28) 32 (40)* 1 (5) 18 (33) 15 (32) hlyA 28 (37) 3 (12) 29 (36) 2 (10) 18 (33) 13 (28) papA 34 (45) 8 (32) 32 (40) 11 (52) 25 (45) 18 (38) papGII 10 (13) 0 9 (11) 1 (5) 6 (11) 4 (9) papGIII 11 (15) 3 (12) 13 (16) 2 (10) 9 (16) 6 (13) sfaDE 33 (44) 4 (16) 33 (41) 4 (19) 24 (44) 13 (28) afa/draBC 1 (1) 0 1 (1) 0 1 (2) 0 iucD 33 (44) 14 (56) 35 (44) 13 (62) 25 (45) 23 (49) kpsMT 52 (69) 13 (52) 53 (66) 12 (57) 36 (65) 30 (64) ompT 64 (85) 17 (68) 69 (86) 12 (57) 48 (87) 34 (72) usp 39 (52) 5 (20) 43 (54)** 2 (10) 26 (47) 19 (40) Number of VFs 0 3 (4) 3 (12) 3 (4) 4 (19) 2 (4) 5 (11) 1 5 (7) 5 (20) 7 (9) 3 (14) 5 (9) 5 (11) 2 7 (9) 4 (16) 9 (11) 1 (5) 6 (11) 5 (11) 3 11 (15) 2 (8) 9 (11) 4 (19) 7 (13) 6 (13) 4 17 (23) 6 (24) 18 (23) 5 (24) 13 (24) 10 (21) 5 14 (19) 3 (12) 14 (18) 4 (19) 8 (15) 10 (21) 6 10 (13) 0 10 (13) 0 8 (15) 2 (4) 7 4 (5) 2 (8) 6 (8) 0 4 (7) 2 (4) 8 4 (5) 0 4 (5) 0 2 (4) 2 (4) Average virulence score 4,1 2,9 4,1 2,7 4,0 3,5
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TABLE 4: Comparison of prevalence of phylogenetic groups and VFs of our and previous studies, performed on ExPEC subgroups, and with previous studies on fecal 503
isolates. 504
Prevalence (no. [%] of isolates)
Present study (no. of isolates)
Previous studies (no. of isolates)
Skin and soft tissue infections
(102)
Meningitisa (70)
Recurrent cystitisb
(74)
First UTI womenc (93)
Vaginad (88) Bacteremiae (63)
UTIf (377) Acute cystitisg (100)
Rectal isolatese
(71) Human fecesh
(266)
Phylogenetic group
A 12 (12) 1 (1)(*) - 7 (8) 7 (8) 7 (11) - 20 (20) 11 (15) 52 (20)
B1 10 (10) 7 (10) - 3 (3) 0(**) 2 (3) - 6 (6) 13 (18) 33 (11)
B2 66 (65) 57 (81) - 64 (69) 67 (76) 42 (67) - 55 (55) 38 (54) 120 (45)(**)
D 14 (14) 5 (7) - 19 (20) 14 (16) 12 (19) - 19 (19) 9 (13) 61 (23) Virulence factor (VF) cnf1 33 (32) 6 (9)(**) 25 (34) 26 (28) 17 (19) 23 (37) - 34 (34) 9 (13)(*) 43 (16)(**) hly 31 (30) 6 (9)(**) 27 (36) 31 (33) 19 (22) 28 (44) - 44 (44) 10 (14) 51 (19) papA 43 (41) - 26 (35) - - - - 46 (46) - -
papGII + GIII 24 (24) 15 (21) 27 (36) - - 43 (68)*** - 48 (48)** 19 (27) -
sfa 37 (36) 41 (59) 23 (31) 26 (41) 18 (20) 8 (13)(*) - 6 (11) (***) 5 (7)(***) -
afa/dra 1 (1) 18 (26)*** 3 (4) 17 (18)*** 5 (6) 3 (5) - 14 (14)** 4 (6) 14 (5) aer (iucD/iutA) 48 (47) 43 (61) 21 (28) 46 (49) 31 (35) 34 (54) - - 14 (20)(**) 86 (32) kpsMT 66 (65) 60 (86)* 46 (62) 76 (82) - 50 (79) 342 (91)*** 69 (69) 34 (48) 142 (53) ompT 82 (80) 67 (96)* - 81 (87) - 51 (81) 354 (94)** 53 (53) (***) 11 (15)(***) 155 (58)(***)
usp 45 (44) - - - - - 320 (85)*** - - - a Ref. (20); b Ref. (18); c Ref. (50); d Ref. (34); e Ref. (39); f Ref. (24); g Ref. (16); h Ref. (11). 505
Statistical significance of differences in prevalence was calculated between our strain collection and every single ExPEC collection presented in the table. P values following 506
Bonferroni correction are indicated by asterisk where P is <0.05. Symbols: *, P<0.05; **,P<0.01; ***, P<0.001. 507
508 509
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JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2010, p. 3462–3463 Vol. 48, No. 90095-1137/10/$12.00 doi:10.1128/JCM.01266-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.
AUTHOR’S CORRECTION
Virulence Potential of Escherichia coli Isolates from Skin and SoftTissue Infections
Ziva Petkovsek, Kristina Elersic, Marija Gubina, Darja Zgur-Bertok, and Marjanca Starcic ErjavecDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia; Department of
Surface Engineering and Optoelectronics, Institut Jozef Stefan, Teslova ulica 30, 1000 Ljubljana, Slovenia; and Institute ofMicrobiology and Immunology, Medical Faculty, University of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia
Volume 47, no. 6, p. 1811–1817, 2009. We recently discovered that when transferring the obtained data of phylogenetic groupsinto the table used for statistical analysis, for some SSTI isolates, incorrect phylogenetic groups were transferred, leading to somemistakes in the published article as follows.
Page 1811, abstract, line 8: “(65%)” should read “(64%).”Page 1812: Table 1 (including footnote a) should read as shown below.
VF or no. of VFs
Prevalence of trait (no. [%] of isolates) by phylogenetic group
All isolates(n � 102[100%])
A (n � 12 [12%]) B1 (n � 9 [9%]) B2 (n � 65 [64%]) D (n � 16 [16%])
Toxinscnf1 33 (32) 1 (8) 2 (22) 28 (43)* 2 (13)hlyA 31 (30) 0 3 (33) 25 (38) 3 (19)
Fimbriae and/or adhesinspapA 43 (41) 3 (25) 0 36 (55)* 4 (25)papGII 10 (10) 0 0 8 (12) 2 (13)papGIII 15 (15) 1 (8) 0 14 (22)* 0sfaDE 37 (36) 1 (8) 4 (44) 29 (45) 3 (19)afa/draBC 1 (1) 0 0 0 1 (6)
Iron uptakeiucD 48 (47) 5 (42) 3 (33) 31 (48) 9 (56)
CapsulekpsMT 66 (65) 1 (8) 4 (44) 53 (82)*** 9 (56)
OtherompT 82 (80) 7 (58) 1 (11) 63 (97)*** 11 (69)usp 45 (44) 0 0 43 (66)*** 2 (13)
No. of VFs0 7 (7) 2 (17) 4 (44) 0 (**) 1 (6)1 10 (10) 4 (33) 1 (11) 1 (2) (**) 4 (25)2 11 (11) 5 (42) 1 (11) 2 (3) (*) 3 (19)3 13 (13) 0 0 12 (18) 1 (6)4 23 (23) 1 (8) 2 (22) 16 (25) 4 (25)5 18 (18) 0 1 (11) 14 (22) 3 (19)6 10 (10) 0 0 10 (15) 07 6 (6) 0 0 6 (9) 08 4 (4) 0 0 4 (6) 0
a A correlation between a VF and the phylogenetic group B2 was ascertained by comparing the rates of prevalence of VFs between B2 and all non-B2 strains. Fisher’sexact test and Bonferroni’s correction were used to analyze the data. The P value after Bonferroni’s correction is indicated by asterisks as follows: *, P � 0.05; **, P �0.01; ***, P � 0.001. Negative correlations are indicated by asterisks in parentheses.
3462
Page 1812, right column, paragraph 1, line 2: “66 strains (65%)” should read “65 strains (64%).”Page 1812, right column, paragraph 1, line 3: “Fourteen isolates (14%)” should read “Sixteen isolates (16%).”Page 1812, right column, paragraph 1, line 4: “10 (10%)” should read “9 (9%).”Page 1813: The first four rows (the “Phylogenetic group” section) and footnote a of Table 2 should read as shown below.
Page 1813, left column, paragraph 3, lines 5 and 6: “although only the association of papA with the B2 group was statisticallysignificant (P � 0.01) (Table 1)” should read “and several statistically significant associations were found, namely, with cnf1, papA,papGIII, kpsMTIII, ompT, and usp (Table 1).”
Page 1813, left column, paragraph 4, line 3: “six to eight VFs” should read “three to eight VFs.”Page 1813, left column, paragraph 4, lines 4 and 5: the sentence “Strains encoding three to five VFs were more equally
distributed among all phylogenetic groups” should be deleted.Page 1814: The first four rows (the “Phylogenetic group” section) and footnote a of Table 3 should read as shown below.
Page 1815: The first four rows (the “Phylogenetic group” section) and footnote i of Table 4 should read as shown below.
Phylogeneticgroup, VF, or
no. of VFs
Prevalence (no. [%] of the tested strains)
Sex Age Infection type Immune system status
Male(n � 58[57%])
Female(n � 44[43%])
�19 yr(n � 19[19%])
19–45 yr(n � 15[15%])
46–65 yr(n � 26[25%])
�65 yr(n � 42[41%])
Acute(n � 63[62%])
Chronic(n � 39[38%])
Normal(n � 49[48%])
Weakened(n � 53[52%])
Phylogeneticgroup
A 10 (17) 2 (5) 3 (16) 1 (7) 5 (19) 3 (7) 10 (16) 2 (5) 7 (14) 5 (9)B1 5 (9) 4 (9) 2 (11) 1 (7) 4 (15) 2 (5) 5 (8) 4 (10) 4 (8) 5 (9)B2 32 (55) 33 (75) 9 (47) 11 (73) 14 (54) 31 (74) 37 (59) 28 (72) 28 (57) 37 (70)D 11 (19) 5 (11) 5 (26) 2 (13) 3 (12) 6 (14) 11 (17) 5 (13) 10 (20) 6 (11)
a A correlation between phylogenetic groups and patient characteristics was ascertained using Fisher’s exact test and Bonferroni’s correction. No statisticallysignificant correlations were found.
Phylogeneticgroup or VF
Prevalence (no. [%] of isolates)
SSTI frompresentstudy
(n � 102)
Sample type from previous study
Meningitisa
(n � 70)
Recurrentcystitisb
(n � 74)
UTIc
(n � 93women)
Vaginad
(n � 88)Bacteremiae
(n � 63)UTIf
(n � 377)
Acutecystitisg
(n � 100)
Rectal isolatese
(n � 71)Human fecesh
(n � 266)
Phylogeneticgroup
A 12 (12) 1 (1) (*) 7 (8) 7 (8) 7 (11) 20 (20) 11 (15) 52 (20)B1 9 (9) 7 (10) 3 (3) 0 (**) 2 (3) 6 (6) 13 (18) 33 (11)B2 65 (64) 57 (81) 64 (69) 67 (76) 42 (67) 55 (55) 38 (54) 120 (45) (**)D 16 (16) 5 (7) 19 (20) 14 (16) 12 (19) 19 (19) 9 (13) 61 (23)
i Statistical significance of differences in prevalence was calculated between our strain collection and every single ExPEC collection presented in the table. P values,determined using Fisher’s exact test and Bonferroni’s correction, are indicated with asterisks: *, P � 0.05; **, P � 0.01. Negative correlations are indicated by asterisksin parentheses.
Phylogenetic group,VF, or no. of VFs
Prevalence (no. [%] of strains)
Tetracycline Ciprofloxacin Ampicillin
Susceptible(n � 75[74%])
Resistant(n � 25[25%])
Susceptible(n � 80[78%])
Resistant(n � 21[21%])
Susceptible(n � 55[54%])
Resistant(n � 47[46%])
Phylogenetic groupA 8 (11) 4 (16) 8 (10) 4 (19) 8 (15) 4 (9)B1 5 (7) 4 (16) 5 (6) 4 (19) 3 (5) 6 (13)B2 54 (72) 11 (44) 53 (66) 12 (57) 36 (65) 29 (62)D 10 (13) 6 (24) 15 (19) 1 (5) 8 (15) 8 (17)
a A correlation between phylogenetic groups and resistance phenotypes was ascertained using Fisher’s exact test and Bonferroni’s correction. No statisticallysignificant correlations were found.
VOL. 48, 2010 AUTHOR’S CORRECTION 3463
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