JCM Accepts, published online ahead of print on 8 April ...€¦ · 20 Ljubljana, Zalo ka 4, 1000...

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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

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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

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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

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* 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: [email protected]

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

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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

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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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122

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

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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

https://crossmark.crossref.org/dialog/?doi=10.1128/JCM.01266-10&domain=pdf&date_stamp=2010-09-01

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%])





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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JCM Accepts, published online ahead of print on 8 April ...€¦ · 20 Ljubljana, Zalo ka 4, 1000...

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