Plasmid-mediated resistance to cephalosporins and ...

1

Plasmid-mediated resistance to cephalosporins and fluoroquinolones in various 1

Escherichia coli sequence types isolated from rooks wintering in Europe 2

3

Ivana Jamborova1#, Monika Dolejska

1,2, Jiri Vojtech

1,2, Sebastian Guenther

3, Raluca 4

Uricariu1, Joanna Drozdowska

1,4, Ivo Papousek

1, Katerina Pasekova

1, Wlodzimierz 5

Meissner4, Jozef Hordowski

5, Alois Cizek

2,6 and Ivan Literak

1,2 6

7

1Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, 8

University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic 9

2CEITEC, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic 10

3Institute of Microbiology and Epizootics, Veterinary Faculty, Free University Berlin, Berlin, 11

Germany 12

4Avian Ecophysiology Unit, Department of Vertebrate Ecology and Zoology, University of 13

Gdańsk, Gdańsk, Poland 14

5Arboretum I Zaklad Fizjografii w Bolestraszycach, Przemysl, Poland 15

6Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, 16

University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic 17

18

#Address correspondence to I. Jamborova, Department of Biology and Wildlife Diseases, 19

Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical 20

Sciences Brno, Palackeho tr. 1/3, 612 42 Brno, Czech Republic. Telephone: +420 541 562 21

644. Fax: +420 541 562 631. E-mail: [email protected] 22

23

Running title: ESBL, AmpC and PMQR E. coli from rooks in Europe 24

Keywords: ESBL, AmpC, PMQR, MLST, wildlife, Corvus frugilegus 25

AEM Accepts, published online ahead of print on 7 November 2014Appl. Environ. Microbiol. doi:10.1128/AEM.02459-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

on April 5, 2018 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

2

Abstract 26

Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC)-producing 27

and plasmid-mediated quinolone-resistant (PMQR) strains of Escherichia coli were 28

investigated in wintering rooks (Corvus frugilegus) from eight European countries. 1073 feces 29

samples from rooks wintering in the Czech Republic, France, Germany, Italy, Poland, Serbia, 30

Spain and Switzerland were examined. Resistant isolates obtained from selective cultivation 31

were screened for ESBL, AmpC and PMQR genes by PCR and sequencing. Pulsed-field gel 32

electrophoresis and multi-locus sequence typing were performed to reveal their clonal 33

relatedness. In total, 152 (14%, nsamples=1073) cefotaxime-resistant E. coli isolates and 355 34

(33%, nsamples=1073) E. coli with reduced susceptibility to ciprofloxacin were found. Eighty-35

two (54%) of these cefotaxime-resistant E. coli isolates carried ESBL genes as follow: blaCTX-36

M-1 (n=39), blaCTX-M-15 (25), blaCTX-M-24 (4), blaTEM-52 (4), blaCTX-M-14 (2), blaCTX-M-55 (2), 37

blaSHV-12 (2), blaCTX-M-8 (1), blaCTX-M-25 (1), blaCTX-M-28 (1), and one not specified. Forty-seven 38

(31%) cefotaxime-resistant E. coli isolates encoded AmpC beta-lactamases blaCMY-2. Sixty-39

two (17%) of E. coli isolates with reduced susceptibility to ciprofloxacin were positive for the 40

PMQR genes qnrS1 (n=54), qnrB19 (4), qnrS1+qnrB19 (2), qnrS2 (1) and aac(6´)-Ib-cr (1). 41

Eleven isolates from the Czech Republic (8) and Serbia (3) were identified as CTX-M-15-42

producing E. coli clone B2-O25b-ST131. Ninety-one different sequence types (ST) among 43

191 ESBL, AmpC and PMQR E. coli isolates were determined, with ST58 (n=15), ST10 (14) 44

and ST131 (12) predominating. Widespread occurrence of highly diverse ESBL-, AmpC- and 45

PMQR-positive E. coli, including the clinically important multi-resistant ST69, ST95, ST117, 46

ST131 and ST405 clones, was demonstrated in rooks wintering in various European 47

countries. 48

49

50


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

3

Introduction 51

The incidence of bacteria resistant to cephalosporins and fluoroquinolones is growing steadily 52

and constitutes a serious risk for human and animal health. The major mechanism conferring 53

resistance to cephalosporins is mediated by extended-spectrum beta-lactamases (ESBLs) and 54

AmpC beta-lactamases (AmpC) (1). Although plasmid-mediated quinolone-resistance 55

(PMQR) genes provide only a low level of resistance to fluoroquinolones and resistance is 56

mainly caused by point mutations of quinolone resistance-determining region (QRDR) coding 57

for gyrase and topoisomerase (2), interaction between mutations in QRDR and PMQR leads 58

to higher levels of resistance to fluoroquinolones (3). 59

Wild animals that do not come directly into contact with antibiotics are affected by their 60

use in human and veterinary medicine. Close proximity of wild animals with humans and 61

domestic animals plays an important role in the transmission of pathogens to wildlife, thus 62

potentially creating an additional environmental reservoir of antibiotic-resistant bacteria (4). 63

Wild birds, and especially corvids, feeding on garbage dumps near urban agglomerations are 64

at high risk of being colonized by multidrug-resistant Escherichia coli isolates. They have 65

been identified as significant carriers and vectors of commensal and pathogenic bacteria with 66

various mechanisms of resistance (4, 5). Probable origin of the majority of wintering rooks in 67

both central and western part of continental Europe is mainly in Eastern Europe including 68

Russia, Belorussia and Ukraine. However, wintering rooks are rare in Italy and Spain 69

nowadays (6). 70

With the implementation of multi-locus sequence typing (MLST) to characterize E. coli 71

and our deeper understanding of microevolution in the core bacterial genome, certain genetic 72

lineages with special features supporting their pandemicity have been described (7, 8). The 73

most studied phylogenetic lineage today in terms of antibiotic resistance is E. coli ST131. 74

This lineage harbors a wide range of virulence genes and various plasmid-mediated resistance 75


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

4

genes, and it is involved in the global spread of the CTX-M-15 beta-lactamase. High level of 76

virulence combined with carriage of transferable elements encoding multi-drug resistance is 77

likely responsible for the pandemic success of ST131 (9). Even as the ST131 clone is known 78

to cause community-onset infections in humans, including urinary tract infections, 79

bacteraemia and neonatal sepsis, it has also been identified in companion animals, poultry, 80

livestock, wild animals and food (10-12). Other lineages with high proportions of multi-drug 81

resistant strains responsible for community-onset and hospital-acquired infections, such as 82

ST69, ST95, ST117 and ST405 (phylogenetic group D), ST10 (A) and ST23 (B1), have been 83

described (13, 14). 84

The findings of particular E. coli lineages in food, water, environment and non-human 85

sources indicate the entirely unexplored complexity of transmission routes (13) whereby 86

migrating birds may play an important role in the circulation of epidemiologically important 87

E. coli clones and may pose a risk of environmental contamination. In this paper, rooks 88

(Corvus frugilegus, a medium-sized corvid) commonly wintering in central and western parts 89

of continental Europe were examined for the presence of ESBL- and AmpC-producing E. coli 90

isolates and/or those carrying PMQR genes. Pulsed-field gel electrophoresis (PFGE) and 91

MLST including eBurst analysis were used to reveal clonal relatedness of these E. coli 92

isolates. This study follows a recent study in European rooks by Literak et al. (2012) where 93

we investigated PMQR genes in pooled samples of Enterobacteriaceae family regardless of 94

bacterial species. 95

96

Methods 97

Collection of rook feces 98

Feces of rooks were collected in nine roosting places in eight European countries during 99

winter (December - February) 2010/2011. An exception was France, where the samples were 100


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

5

collected in April 2011. Fresh feces were picked up individually from plastic film exposed 101

overnight on the ground beneath the roosting place using sterile cotton swab, dipped in Amies 102

Transport Medium (Dispolab, Czech Republic), then transported at room temperature to the 103

laboratory (6). All feces samples were obtained from one sampling site per country except for 104

Germany and Poland. A total of 1073 feces samples from the Czech Republic (150 feces 105

samples), France (31), three sampling sites in Germany (Schortens-Heidmühle, 53°23' N, 106

7°58' E, 54 samples; Wilhelmshaven, 53°32' N, 8°04' E, 33 samples; Schortens-Heidmühle, 107

Huntsteert, 53°32' N, 7°55' E, 13 samples), Italy (145), two locations in Poland (Gdynia, 150 108

samples; Jaroslaw, 148 samples), Serbia (150), Spain (150) and Switzerland (49) were 109

collected. Description of the location and exact time of sampling has been described in our 110

previous study (6). 111

112

Isolation and determination of E. coli producing ESBL, AmpC or carrying PMQR genes 113

Fecal samples were transferred from Amies Transport Medium to buffered peptone water 114

(Oxoid, UK) and cultivated overnight. Subsequently, samples were subcultivated on 115

MacConkey agar (MCA) containing cefotaxime (2 mg/l) and in parallel on MCA with 116

ciprofloxacin (0.05 mg/l). Isolated coliform colonies were identified by matrix-assisted laser 117

desorption ionisation – time-of-flight mass spectrometry (MALDI-TOF; MALDI Biotyper, 118

Bruker Daltonics, USA). The colonies grown on MCA supplemented with cefotaxime were 119

tested for production of ESBL using double-disc synergy test (15). Simultaneously, the 120

isolates were screened for AmpC using cefoxitin (30 µg) disk; those showing cut-off value 121

≤18 mm were confirmed as AmpC producers by cefoxitin-cloxacillin CC-DDS method (16), 122

where cloxacillin have been adequately replaced by oxacilin (128 mg/l) (17). AmpC 123

phenotype in isolates positive by CC-DDS method but negative for all AmpC genes tested 124

was confirmed using MASTDISCSTM

ID AmpC and ESBL test (Mast Diagnostics, 125


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

6

Merseyside, UK). Each ESBL-producing isolate was screened by PCR for the following 126

resistance genes responsible for the ESBL phenotype: blaCTX-M, blaTEM, blaOXA and blaSHV 127

(18). Specific primers for determination of CTX-M subgroups were used (19, 20). AmpC 128

genes blaDHA, blaACC-1, blaACC-2, blaMOX, blaCMY and blaFOX were identified by PCR (21). 129

Sequence type of blaCMY gene was determined using forward primer CMY 2/F (5´-130

AACACACTGATTGCGTCT-3´) and revers primer CMY 2/R (5´-131

CTGGGCCTCATCGTCAGT-3´) based on the reference sequence HQ680723. The colonies 132

grown on MCA with ciprofloxacin were investigated for PMQR genes qnrA, qnrB, qnrC, 133

qnrD, qnrS, aac(6′)-Ib-cr, qepA and oqxAB (18). All PCR products of ESBL, AmpC and 134

PMQR genes were analysed by sequencing. 135

136

Antibiotic susceptibility testing 137

ESBL and PMQR-producing E. coli isolates were tested using disc diffusion method for 138

susceptibility to 13 antimicrobial agents as follows: amoxicillin-clavulanic acid (30 µg), 139

ampicillin (10 µg), cephalothin (30 µg), ceftazidime (30 µg), chloramphenicol (30 µg), 140

ciprofloxacin (5 µg), gentamicin (10 µg), nalidixic acid (30 µg), streptomycin (10 µg), 141

sulfamethoxazole-trimethoprim (25 µg), sulfonamide compounds (300 µg), tetracycline (30 142

µg) and imipenem (10 µg) according to CLSI guidelines (15, 22). 143

144

Molecular typing methods 145

Epidemiological relatedness of E. coli was detected by XbaI PFGE and MLST with an 146

MSTree (23). Macrorestriction patterns and sequence type complexes in the MSTree were 147

calculated using BioNumerics 6.6 (Applied Maths, Ghent, Belgium). Cluster analysis of the 148

Dice similarity indices was done to generate a dendrogram describing the relationships among 149


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

7

PFGE profiles. The minimum level for similarity between patterns was defined to be 85% 150

(24). 151

PCR for MLST was based on seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA 152

and recA). Different sequences of a given locus were assigned allele numbers based on the E. 153

coli MLST database (http://mlst.warwick.ac.uk/mlst/), and each unique combination of alleles 154

(the allelic profile) was determined as a sequence type (ST) (7). The eBURST 155

(http://eburst.mlst.net/) algorithm was used for determining clonal complexes of STs (STCs), 156

whereby STs sharing six or more loci were assigned to defined STCs (25). Phylogenetic type 157

affiliation was determined using Structure analysis based on seven housekeeping genes 158

(http://pritch.bsd.uchicago.edu/structure) and for six isolates by multiplex PCR assay (26). 159

160

Results 161

ESBL, AmpC and PMQR genes in E. coli isolates 162

A total of 152 (14%, nsamples=1073) cefotaxime-resistant E. coli isolates were found. Eighty-163

two (54%, nE. coli =152) of these cefotaxime-resistant E. coli isolates carried an ESBL gene 164

and none of them harbored AmpC gene. The gene blaCTX-M-1 was predominant (n=39), 165

followed by blaCTX-M-15 (25). The other ESBL genes were rare and more locally distributed 166

(Tables 1 and 2). The ESBL-producing isolates belonged to phylogenetic groups A (32 167

isolates, 39%, n=82), B1 (2, 2%), B2 (12, 15%), A×B1 (20, 24%) and ABD (16, 20%). 168

Genetic background of hybrid group A×B1 is more likely from their ancestry groups A and 169

B1, while ABD is more diverse group with multiple sources of ancestry. Due to extensive 170

recombination within these two hybrid groups, higher proportion of pathogens is assigned 171

into these groups (7). Twenty-two (27%, nESBL=82) isolates with ESBL phenotype also 172

carried the gene aac(6´)-Ib-cr responsible for resistance to fluoroquinolones and 173

aminoglycosides. Thirty-one (38%) and 17 isolates (21%) also carried blaTEM-1 and blaOXA-1, 174


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

8

respectively. In Germany, two SHV-12-producing isolates also carried the qnrS1 gene (Table 175

S1 available as Supplemental material at AEM online). 176

Fifty-six (37%, nE. coli =152) of cefotaxime-resistant E. coli isolates displayed AmpC 177

phenotype. The gene blaCMY-2 was the only detected AmpC gene identified in forty-seven 178

(31%, nE. coli =152) isolates (Table 1 and 3). CMY-2-producing E. coli isolates were members 179

of phylogenetic groups A (9 isolates, 19%, n=47), B1 (3, 6%), B2 (5, 11%), D (6, 13%), 180

A×B1 (8, 17%) and ABD (15, 32%) and one not specified. Ten isolates (21%, n=47) also 181

carried blaTEM-1 and the gene qnrB5/19 was found in one isolate. One isolate from Poland, 182

Gdynia carried qnrS1 and blaTEM-135 genes (Table S2). 183

A total of 355 (33%, nsamples=1073) E. coli isolates were obtained by selective cultivation 184

on media with criprofloxacin. Sixty-two (17%, nE. coli =355) of these isolates carried PMQR 185

genes, the qnrS1 gene being the most frequent (found in 54 isolates from 5 countries). Other 186

isolates carried qnrB19 or aac(6´)-Ib-cr genes (Tables 1 and 4). Isolates with PMQR genes 187

belonged to phylogenetic groups A (25 isolates, 40%, n=62), B1 (9, 15%), A×B1 (18, 29%) 188

and ABD (10, 16%). Forty-six isolates (74%) also carried blaTEM-1, and 7 isolates (11%) from 189

a polish locality contained blaTEM-32 (Table S3). 190

191

Antibiotic resistance phenotypes 192

Almost all, 66 out of 82 ESBL-producing isolates (80%) were multiresistant (resistant 193

to three or more antibiotic groups); (Table S1). ESBL-positive E. coli isolates showed 194

resistance to sulphonamides (76%), tetracycline (61%), trimethoprim-sulphamethoxazole 195

(57%), nalidixic acid (52%), ciprofloxacin (44%), streptomycin (27%), gentamicin (23%), 196

amoxicillin-clavulanic acid (16%), ceftazidime (16%) and chloramphenicol (7%). ESBL-197

producing E. coli with high resistance to 7- 11 antimicrobial agents were observed on the 198

Czech and Serbia localities due to the presence of multidrug-resistant successful clones and 199


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

9

clonal complexes ST131 (11), STC10 (7), ST3014 (2) and ST405 (1) which showed 200

resistance to 7-9 antimicrobial agents. On the other localities, isolates with high resistance 201

rate to 7-10 antimicrobial agents were distributed among the predominant clonal complexes 202

found in this study STC10 (5), STC155 (2), STC23 (1) and only sporadically among other 203

minor complexes or ST types (not exceeding one) were found. 204

All AmpC positive isolates were multiresistant (Table S2) and showed resistance to 205

ceftazidime (79%), nalidixic acid (51%), sulphonamides (36%), tetracycline (34%), 206

streptomycin (30%), trimethoprim-sulphamethoxazole (28%), ciprofloxacin (23%), 207

chloramphenicol (15%) and gentamicin (6%). Eleven out of 18 AmpC producing E. coli 208

isolates in Poland, Gdynia showed high level of resistance to 7-11 antimicrobial agents and 209

belonged mainly to complexes STC86 (3), STC10 (2) and STC155 (2). Only five isolates 210

(n=27) with AmpC beta-lactames were highly multiresistant (7-11 antimicrobial agents) in the 211

Czech Republic and showed various STs. 212

A total of 27 (44%) PMQR-positive isolates were multiresistant (Table S3). PMQR-213

positive E. coli isolates showed resistance to ampicillin (85%), tetracycline (52%), nalidixic 214

acid (35%), streptomycin (32%) sulphonamides (24%), trimethoprim-sulphamethoxazole 215

(21%), amoxicillin-clavulanic acid (13%), ciprofloxacin (8%), chloramphenicol (6%) and 216

gentamicin (2%). The isolates showing resistance up to 7-9 antimicrobial agents belonged to 217

three dominant clonal complexes found in this study STC10 (2), STC23 (2) and STC155 (2). 218

None of the PMQR-positive E. coli isolates selected on media with ciprofloxacin were ESBL 219

or AmpC producers. 220

221

Clonal similarity and MLST of E. coli carrying ESBL, AmpC or PMQR genes 222

Overall, the isolates tested by PFGE showed very high variability in all localities except 223

ESBL-producing strains in Czech Republic (Figure S1, S2, S3; available as Supplemental at 224


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

10

AEM online). Significant clonal similarity was demonstrated in the Czech locality where 225

three clusters, out of 16 among 38 ESBL-producing strains represented by ST types ST131 (8, 226

21%), ST58 (7, 18%) and ST617 (5, 13%) were most frequently identified. ST131 isolates 227

were assigned to one cluster based on less stringent criteria (Dice similarity index of their 228

macrorestriction profiles ≥79.6%). MLST types in Germany and Poland, Gdynia, were more 229

variable, with ST10 in Germany and ST115 forming one cluster in Poland, Gdynia 230

predominating (Figure S1,). Eight novel E. coli STs (Table 2) producing an ESBL were 231

identified in our study. No significant clonal similarity between isolates carrying AmpC or 232

PMQR genes was found in any location (Figure S2 and S3). In the Czech Republic, cluster 233

represented by ST351 (n=4) with blaCMY-2 and ST48 (n=4) harboring the qnrS1 gene were 234

predominant. Identical pulsotypes were found in each locality, but only in minimal amounts 235

(not exceeding four). Three and six novel E. coli STs (Table 3 and 4) with AmpC and PMQR 236

genes, respectively, were identified. Six E. coli isolates were non-typeable with XbaI PFGE 237

(Figure S1 and S3). 238

Overall, 91 different STs were determined among 191 ESBL-, AmpC-producing and 239

PMQR E. coli strains from 9 rook roosting places in 8 European countries, with ST58 (15), 240

ST10 (n=14) and ST131 (12) and as predominant types (Table 2, 3, 4). Thirty-seven percent 241

(71 isolates, n=191) of the isolates belonged to clonal complex STC10. STC155 was the 242

second-largest detected complex, including 33 isolates (17%). STC23 consisted of 8 isolates 243

(4%). Genetic relatedness within each clonal complex can be seen in Figure 1 (based on data 244

from the MLST database (http://mlst.warwick.ac.uk/mlst/, accessed 23 July 2014). 245

Twelve isolates from the Czech Republic (8), Serbia (3) and Poland, Jaroslaw (1) were 246

identified as pandemic multi-resistant E. coli clone B2-O25b-ST131. Eleven ST131 isolates 247

were positive for the gene blaCTX-M-15 and showed closely related PFGE profiles (defined by 248

79.6% band similarity). Six isolates carried all three resistance genes (blaTEM-1, blaOXA-1, 249


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

11

aac(6´)-Ib-cr) typical for this pandemic clone; moreover, in two isolates the plasmid-mediated 250

quinolone-resistance gene qnrB1 gene was detected. Remaining six ST131 isolates carried the 251

blaOXA-1 and aac(6´)-Ib-cr genes. One ST131 isolate from Poland, Jaroslaw carried blaCMY-2 252

gene. 253

According to the MLST, other important sequence types have been found in our study. 254

Four ST69 isolates from Czech Republic (n=3) and Poland, Jaroslaw (1) carried blaCMY-2 (2) 255

and qnrS1 (2) genes (Table 3, 4). ST95 (1) from Poland, Jaroslaw and ST117 (3) from the 256

Czech Republic (1) and Poland, Gdynia (2) encoded AmpC beta-lactamase CMY-2 (Table 3). 257

One blaCTX-M-15 producing isolate from the Czech Republic belonged to ST405 (Table 2). 258

259

Discussion 260

The most predominant ESBL gene in our study was blaCTX-M-1 (n=39; 48%), followed by 261

blaCTX-M-15 (25; 30%). Distributions of other ESBL was rare and local. We assume that both 262

humans and domestic animals are likely the sources of the ESBL genes found in the rook E. 263

coli isolates. CTX-M-14 and CTX-M-15 are the major ESBL types in human strains 264

worldwide (11, 13). On the other hand, the most frequently reported ESBLs in European 265

animal isolates are CTX-M-1 followed by CTX-M-14, TEM-52 and SHV-12 (1). CTX-M-1 is 266

the most predominant ESBL type in isolates from companion and food-producing animals and 267

to a lesser extent occurs in humans (11). CTX-M-24 in rook isolates from the Czech Republic 268

and Poland constitutes an interesting finding, since this ESBL type is generally restricted to 269

Asia (27) and reported only sporadically in other parts of the world (28). The highest 270

prevalence of rooks colonized by ESBL-producers (n=38; 25.3%) was at the locality in the 271

Czech Republic. This sampling site is located in the woods of National Nature Reserve, 272

surrounded by fields, agricultural production and urban agglomerations with a waste water 273

treatment plant and garbage dumps nearby. The distribution of ESBL genes closely 274


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

12

corresponded with the situation previously described in hospital facilities (29, 30), domestic 275

animals (31) and the environment including wild birds and wastewater treatment plant 276

effluent (5, 32, 33) within that country. In Germany, highest distribution of ESBL-producing 277

E. coli was found on two sampling sites. In the rural area with high level of agriculture, the 278

gene blaCTX-M-1 was predominant (60%), while the second location, an urban area nearby a 279

hospital clinic, showed high proportion of blaCTX-M-15 (50%) and blaSHV-12 (25%). On the other 280

hand, localities in Spain, Italy and France with known high percentage of ESBL-producing 281

strains in humans (34) showed very low (0.0–0.3%) prevalence of ESBLs in E. coli from 282

rooks. Our results likely reflect lower levels of environmental contamination by ESBL-283

producing bacteria in the specific localities near the examined roosting places. 284

The only detected AmpC beta-lactamase in our study was CMY-2 found in E. coli isolates 285

from the Czech Republic and Poland. CMY-2 is the most common type of AmpC enzymes 286

among Enterobacteriaceae in farm and companion animals, food products as well as in 287

humans. Other types of AmpC beta-lactamases are scarcely reported (1, 11). It has been 288

suggested that poultry can be important reservoir of CMY-2 AmpC beta-lactamases (1). 289

The most predominant PMQR gene among E. coli found in our study was qnrS1. Other 290

PMQR genes qnrS2, qnrB19 and aac(6´)-Ib-cr were less frequent and showed local 291

distribution.. The most affected locations with noticeable colonization of rooks by PMQR-292

harboring E. coli isolates were the roosting places in the Czech Republic and Poland. The 293

occurrence of qnrS genes in E. coli from wild water birds has been previously described in the 294

Czech Republic and on the Baltic Sea coast of Poland (5). High prevalence of the qnrS1 gene 295

among Salmonella spp. and E. coli isolates from domestic animals, food and the environment 296

has been reported in Germany, Italy, Poland and Spain (35). Based on a recent study from the 297

Czech Republic, broilers could be considered as a source of qnrS1- and qnrB19-harbouring E. 298

coli (36). The examined roosting place in the Czech Republic is surrounded by poultry farms 299


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

13

that spread the bedding material onto the fields as fertilizer and likely serve as a source of 300

PMQR bacteria for the environment where the rooks seek food. Rooks from night roost in 301

Gdynia (Poland) use both urbanized areas and municipal rubbish dump as foraging sites. 302

Hence, these birds may have been in contact with different sources of these genes from 303

human environment. 304

PFGE analysis and MLST demonstrated significant clonal similarity only among the 305

ESBL-producing isolates in the Czech location. Notably, more than one-third of all ESBL- 306

and AmpC-producing isolates belonged to the extraintestinal pathogenic E. coli (ExPEC)-307

linked phylogenetic groups B2 and hybrid group ABD, which contains, among others, 308

internationally successful ST types such as ST69, ST95, ST117 and ST405 (7, 8, 14). It 309

seems, that these two hybrid groups A×B1 and ABD comprised isolates with high frequency 310

of recombination and therefore contain pathogens more frequently (7). Antibiotic-resistant 311

ST69 is a lineage highly associated with UTIs that is disseminated worldwide (13, 14). ST95 312

is prominent, highly invasive and virulent pathogen responsible for human and avian 313

infections (12, 14). ST405 has been described worldwide as a producer of various CTX-M 314

types and is also associated with New Delhi metallo (NDM)-β-lactamases and OXA-48 (8). 315

The main representative of phylogroup B2 in our collection is an internationally disseminated 316

uropathogenic clone O25:H4-ST131 producing CTX-M-15. In the Czech Republic and 317

Serbia, high proportions of the rook isolates belonged to the sequence type ST131. In the 318

Czech Republic, the ST131 clone has been recorded in hospital-onset infections and 319

wastewaters (29, 30, 32). On the other hand, PMQR-positive strains belonged almost 320

exclusively to phylogenetic groups A, B1 and A×B1 (84%) and only sporadically to other 321

groups. 322

Although STs identified in our study were diverse where 91 different ST types among 191 323

ESBL- and AmpC -producing and PMQR E. coli strains were found, 37% of E. coli strains 324


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

14

belonged or were closely related to STC10, followed by clonal complexes STC155 and 325

STC23. STC10 includes pathogenic heterogeneous enteroaggregative E. coli (EAEC), 326

enterotoxigenic E. coli (ETEC), ExPEC, as well as commensal E. coli strains found in 327

humans and food-producing animals (14, 37, 38). STC10 members contributing to the spread 328

of various ESBL and AmpC genes are widely described in relation to hospital-onset 329

infections (39, 40) but they have have been also reported in food animals in the Netherlands, 330

UK and France (41-43). The two main representatives of STC155 in our study were ST58 and 331

ST155. CTX-M-producing STC155 is closely linked to urinary tract infections (UTIs); (44) 332

and it has been reported recently from Italy, Spain, Israel (40). Moreover, the STC155 333

members were associated with various infections in livestock in Europe (42, 43). ESBL-334

producing STC23 is commonly isolated in hospitals in Spain and France (45, 46), but this 335

clonal complex has been also detected in samples obtained from food-producing animals and 336

chicken meat (38, 41, 42). 337

Our study demonstrates high occurrence of common STs or their single and double locus 338

variant known as EAEC, ExPEC and ETEC or previously associated with UTIs. Identical 339

ESBL and/or PMQR genes and E. coli clonal lineages as those found in rooks have been 340

previously reported in humans and domestic animals, thus indicating the plausible sources of 341

the antibiotic-resistant bacteria for rooks. Finding of these bacteria in wildlife likely reflects 342

the presence of such isolates in the birds’ food and water sources as a result of inadequate 343

decontamination procedures regarding wastes of various origins. Based on the MLST 344

database (http://mlst.warwick.ac.uk/mlst/), most STs found in rooks are important human and 345

animal pathogens; their common presence in the environment, including the wildlife, is 346

alarming and should be taken as a serious environmental health risk. Wintering rooks 347

inhabiting urban, suburban and agriculture areas may contribute to the further dissemination 348

of clinically important multi-resistant E. coli clones. 349


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

15

350

Acknowledgements 351

This work was supported by Grant No. NT/14398 from the Ministry of Health of the Czech 352

Republic, the project ‘CEITEC - Central European Institute of Technology’ 353

(CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund. MD and IP were 354

suppostedby The Operational Programme “Education for Competitiveness” 355

(CZ.1.07/2.3.00/30.0014) from the European Social Fund. 356

We thank Frank Borleis, Francisco de la Calzada, Lisa Guardone, Dragan Fabijan, Sebastian 357

Franco, Susanne Homma, Ruben Gonzales Janez, Jiri Klimes, Adam Konecny, Cyrille Lejas, 358

Benito Fuertes Marcos, Veronika Oravcova, Jakub Prochazka, Tomas Lang, Simona 359

Krepelova, Zuzana Markova, Hana Dobiasova, Radim Petro, Marko Sciban, Marie Slavikova, 360

Eva Suchanova and Marko Tucakov for excellent cooperation in the field or in the laboratory. 361

Our special thanks go to Lars Hansen (University of Copenhagen, Denmark), Lina Cavaco 362

and Henrik Hasman (National Food Institute, Copenhagen, Denmark) and Lothar H. Wieler 363

and Torsten Semmler (Free University, Berlin, Germany) for control strains and for advice on 364

methodologies. 365

366

References: 367

1. EFSA Panel on Biological Hazards (BIOHAZ). 2011. Scientific Opinion on the 368

public health risks of bacterial strains producing extended-spectrum beta-lactamases 369

and/or AmpC beta-lactamases in food and food-producing animals. EFSA Journal 9: 370

2322. doi:10.2903/j.efsa.2011.2322. Available online: 371

http://www.efsa.europa.eu/en/publications/efsajournal.htm (1 January 2014, date last 372

accessed). 373


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

16

2. Hopkins KL, Davies RH, Threlfall EJ. 2005. Mechanisms of quinolone resistance in 374

Escherichia coli and Salmonella: recent developments. International Journal of 375

Antimicrobial Agents 25:358-373. 376

3. Luo YP, Li JY, Meng Y, Ma Y, Hu CQ, Jin SH, Zhang QS, Ding H, Cui SH. 377

2011. Joint effects of topoisomerase alterations and plasmid-mediated quinolone-378

resistant determinants in Salmonella enterica Typhimurium. Microbial Drug 379

Resistance 17:1-5. 380

4. Guenther S, Ewers C, Wieler LH. 2011. Extended-spectrum beta-lactamases 381

producing E. coli in wildlife, yet another form of environmental pollution? Front 382

Microbiol 2:246. 383

5. Literak I, Dolejska M, Janoszowska D, Hrusakova J, Meissner W, Rzyska H, 384

Bzoma S, Cizek A. 2010. Antibiotic-resistant Escherichia coli bacteria, including 385

strains with genes encoding the extended-spectrum beta-lactamase and QnrS, in 386

waterbirds on the Baltic Sea Coast of Poland. Applied and Environmental 387

Microbiology 76:8126-8134. 388

6. Literak I, Micudova M, Tausova D, Cizek A, Dolejska M, Papousek I, Prochazka 389

J, Vojtech J, Borleis F, Guardone L, Guenther S, Hordowski J, Lejas C, Meissner 390

W, Marcos BF, Tucakov M. 2012. Plasmid-mediated quinolone resistance genes in 391

fecal bacteria from rooks commonly wintering throughout Europe. Microbial Drug 392

Resistance 18:567-573. 393

7. Wirth T, Falush D, Lan RT, Colles F, Mensa P, Wieler LH, Karch H, Reeves PR, 394

Maiden MCJ, Ochman H, Achtman M. 2006. Sex and virulence in Escherichia coli: 395

an evolutionary perspective. Molecular Microbiology 60:1136-1151. 396

8. Pitout JD. 2012. Extraintestinal pathogenic Escherichia coli: a combination of 397

virulence with antibiotic resistance. Front Microbiol 3:9. 398


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

17

9. Johnson JR, Johnston B, Clabots C, Kuskowski MA, Castanheira M. 2010. 399

Escherichia coli sequence type ST131 as the major cause of serious multidrug-400

resistant E. coli infections in the United States. Clinical Infectious Diseases 51:286-401

294. 402

10. Platell JL, Johnson JR, Cobbold RN, Trott DJ. 2011. Multidrug-resistant 403

extraintestinal pathogenic Escherichia coli of sequence type ST131 in animals and 404

foods. Veterinary Microbiology 153:99-108. 405

11. Ewers C, Bethe A, Semmler T, Guenther S, Wieler LH. 2012. Extended-spectrum 406

beta-lactamase-producing and AmpC-producing Escherichia coli from livestock and 407

companion animals, and their putative impact on public health: a global perspective. 408

Clinical Microbiology and Infection 18:646-655. 409

12. Guenther S, Grobbel M, Beutlich J, Guerra B, Ulrich RG, Wieler LH, Ewers C. 410

2010. Detection of pandemic B2-O25-ST131 Escherichia coli harbouring the CTX-M-411

9 extended-spectrum beta-lactamase type in a feral urban brown rat (Rattus 412

norvegicus). Journal of Antimicrobial Chemotherapy 65:582-584. 413

13. Woodford N, Turton JF, Livermore DM. 2011. Multiresistant Gram-negative 414

bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. Fems 415

Microbiology Reviews 35:736-755. 416

14. Manges AR, Johnson JR. 2012. Food-borne origins of Escherichia coli causing 417

extraintestinal infections. Clinical Infectious Diseases 55:712-719. 418

15. Clinical and Laboratory Standards Institute. 2008. Performance standards for 419

antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 420

Approved Standard CLSI Document M31-A3, 3rd

edition. Clinical and Laboratory 421

Standards Institute, Wayne, PA. 422


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

18

16. Polsfuss S, Bloemberg GV, Giger J, Meyer V, Boettger EC, Hombach M. 2011. 423

Practical approach for reliable detection of AmpC beta-lactamase-producing 424

Enterobacteriaceae. Journal of Clinical Microbiology 49:2798-2803. 425

17. Drieux L, Brossier F, Sougakoff W, Jarlier V. 2008. Phenotypic detection of 426

extended-spectrum beta-lactamase production in Enterobacteriaceae: review and 427

bench guide. Clinical Microbiology and Infection 14:90-103. 428

18. Dobiasova H, Dolejska M, Jamborova I, Brhelova E, Blazkova L, Papousek I, 429

Kozlova M, Klimes J, Cizek A, Literak I. 2013. Extended spectrum beta-lactamase 430

and fluoroquinolone resistance genes and plasmids among Escherichia coli isolates 431

from zoo animals, Czech Republic. Fems Microbiology Ecology 85:604-611. 432

19. Galas M, Decousser J-W, Breton N, Godard T, Allouch PY, Pina P, Col BVHSG. 433

2008. Nationwide study of the prevalence, characteristics, and molecular 434

epidemiology of extended-spectrum-β-lactamase-producing Enterobacteriaceae in 435

France. Antimicrobial Agents and Chemotherapy 52:786-789. 436

20. Jeong SH, Bae IK, Kwon SB, Lee JH, Song JS, Jung HI, Sung KH, Jang SJ, Lee 437

SH. 2005. Dissemination of transferable CTX-M-type extended-spectrum β-438

lactamase-producing Escherichia coli in Korea. Journal of Applied Microbiology 439

98:921-927. 440

21. Perez-Perez FJ, Hanson ND. 2002. Detection of plasmid-mediated AmpC β-441

lactamase genes in clinical isolates by using multiplex PCR. Journal of Clinical 442


22. Clinical and Laboratory Standards Institute. 2011. Performance standards for 444

antimicrobial susceptibility testing; twenty-first informational supplement. CLSI 445

M100-S21. Clinical and Laboratory Standards Institute, Wayne, PA. 446


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

19

23. CDC. 2004. Standardized molecular subtyping of foodborne bacterial pathogens by 447

pulse-field gel electrophoresis. Centers for Disease Control, Atlanta, GA. 448

24. Carrico JA, Pinto FR, Simas C, Nunes S, Sousa NG, Frazao N, de Lencastre H, 449

Almeida JS. 2005. Assessment of band-based similarity coefficients for automatic 450

type and subtype classification of microbial isolates analyzed by pulsed-field gel 451

electrophoresis. Journal of Clinical Microbiology 43:5483-5490. 452

25. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. 2004. eBURST: Inferring 453

patterns of evolutionary descent among clusters of related bacterial genotypes from 454

multilocus sequence typing data. Journal of Bacteriology 186:1518-1530. 455

26. Clermont O, Bonacorsi S, Bingen E. 2000. Rapid and simple determination of the 456

Escherichia coli phylogenetic group. Applied and Environmental Microbiology 457

66:4555-4558. 458

27. Nguyen TKN, Ha V, Tran VTN, Stabler R, Pham TD, Le TMV, van Doorn HR, 459

Cerdeno-Tarraga A, Thomson N, Campbell J, Nguyen VMH, Tran TTN, Pham 460

VM, Cao TT, Wren B, Farrar J, Baker S. 2010. The sudden dominance of bla(CTX-461

M) harbouring plasmids in Shigella spp. circulating in Southern Vietnam. Plos 462

Neglected Tropical Diseases 4. 463

28. Pitout JDD, Gregson DB, Campbell L, Laupland KB. 2009. Molecular 464

characteristics of extended-spectrum-beta-lactamase-producing Escherichia coli 465

isolates causing bacteremia in the Calgary Health Region from 2000 to 2007: 466

emergence of clone ST131 as a cause of community-acquired infections. 467

Antimicrobial Agents and Chemotherapy 53:2846-2851. 468

29. Dolejska M, Brhelova E, Dobiasova H, Krivdova J, Jurankova J, Sevcikova A, 469

Dubska L, Literak I, Cizek A, Vavrina M, Kutnikova L, Sterba J. 2012. 470

Dissemination of IncFII(K)-type plasmids in multiresistant CTX-M-15-producing 471


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

20

Enterobacteriaceae isolates from children in hospital paediatric oncology wards. 472

International Journal of Antimicrobial Agents 40:510-515. 473

30. Hrabak J, Empel J, Bergerova T, Fajfrlik K, Urbaskova P, Kern-Zdanowicz I, 474

Hryniewicz W, Gniadkowski M. 2009. International clones of Klebsiella 475

pneumoniae and Escherichia coli with extended-spectrum beta-lactamases in a Czech 476

hospital. Journal of Clinical Microbiology 47:3353-3357. 477

31. Dolejska M, Matulova M, Kohoutova L, Literak I, Bardon J, Cizek A. 2011. 478

Extended-spectrum beta-lactamase-producing Escherichia coli in turkey meat 479

production farms in the Czech Republic: National survey reveals widespread isolates 480

with bla(SHV-12) genes on IncFII plasmids. Letters in Applied Microbiology 53:271-481

277. 482

32. Dolejska M, Frolkova P, Florek M, Jamborova I, Purgertova M, Kutilova I, 483

Cizek A, Guenther S, Literak I. 2011. CTX-M-15-producing Escherichia coli clone 484

B2-O25b-ST131 and Klebsiella spp. isolates in municipal wastewater treatment plant 485

effluents. Journal of Antimicrobial Chemotherapy 66:2784-2790. 486

33. Tausova D, Dolejska M, Cizek A, Hanusova L, Hrusakova J, Svoboda O, Camlik 487

G, Literak I. 2012. Escherichia coli with extended-spectrum beta-lactamase and 488

plasmid-mediated quinolone resistance genes in great cormorants and mallards in 489

Central Europe. Journal of Antimicrobial Chemotherapy 67:1103-1107. 490

34. European Centre for Disease Prevention and Control. 2013. Annual 491

Epidemiological Report 2013. Reporting on 2011 surveillance data and 2012 epidemic 492

intelligence data. Stockholm, ECDC. doi 10.2900/13174. Available online: 493

http://www.ecdc.europa.eu/en/publications/_layouts/forms/Publication_DispForm.asp494

x?List=4f55ad51-4aed-4d32-b960-af70113dbb90&ID=989 495


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

21

35. Veldman K, Cavaco LM, Mevius D, Battisti A, Franco A, Botteldoorn N, 496

Bruneau M, Perrin-Guyomard A, Cerny T, Escobar CD, Guerra B, Schroeter A, 497

Gutierrez M, Hopkins K, Myllyniemi AL, Sunde M, Wasyl D, Aarestrup FM. 498

2011. International collaborative study on the occurrence of plasmid-mediated 499

quinolone resistance in Salmonella enterica and Escherichia coli isolated from 500

animals, humans, food and the environment in 13 European countries. Journal of 501

Antimicrobial Chemotherapy 66:1278-1286. 502

36. Literak I, Reitschmied T, Bujnakova D, Dolejska M, Cizek A, Bardon J, 503

Pokludova L, Alexa P, Halova D, Jamborova I. 2013. Broilers as a source of 504

quinolone-resistant and extraintestinal pathogenic Escherichia coli in the Czech 505

Republic. Microbial Drug Resistance 19:57-63. 506

37. Olesen B, Scheutz F, Andersen RL, Menard M, Boisen N, Johnston B, Hansen 507

DS, Krogfelt KA, Nataro JP, Johnson JR. 2012. Enteroaggregative Escherichia coli 508

O78: H10, the cause of an outbreak of urinary tract infection. Journal of Clinical 509


38. Shepard SM, Danzeisen JL, Isaacson RE, Seemann T, Achtman M, Johnson TJ. 511

2012. Genome sequences and phylogenetic analysis of K88-and F18-positive porcine 512

enterotoxigenic Escherichia coli. Journal of Bacteriology 194:395-405. 513

39. Peirano G, van der Bij AK, Gregson DB, Pitout JDD. 2012. Molecular 514

epidemiology over an 11-year period (2000 to 2010) of extended-spectrum beta-515

lactamase-producing Escherichia coli causing bacteremia in a centralized Canadian 516

region. Journal of Clinical Microbiology 50:294-299. 517

40. Izdebski R, Baraniak A, Fiett J, Adler A, Kazma M, Salomon J, Lawrence C, 518

Rossini A, Salvia A, Samso JV, Fierro J, Paul M, Lerman Y, Malhotra-Kumar S, 519

Lammens C, Goossens H, Hryniewicz W, Brun-Buisson C, Carmeli Y, 520


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

22

Gniadkowski M, Grp MWS, Grp WPS. 2013. Clonal structure, extended-spectrum 521

beta-lactamases, and acquired AmpC-type cephalosporinases of Escherichia coli 522

populations colonizing patients in rehabilitation centers in four countries. 523

Antimicrobial Agents and Chemotherapy 57:309-316. 524

41. Stuart JC, van den Munckhof T, Voets G, Scharringa J, Fluit A, Leverstein-Van 525

Hall M. 2012. Comparison of ESBL contamination in organic and conventional retail 526

chicken meat. International Journal of Food Microbiology 154:212-214. 527

42. Wu GH, Ehricht R, Mafura M, Stokes M, Smith N, Pritchard GC, Woodward 528

MJ. 2012. Escherichia coli isolates from extraintestinal organs of livestock animals 529

harbour diverse virulence genes and belong to multiple genetic lineages. Veterinary 530


43. Dahmen S, Metayer V, Gay E, Madec JY, Haenni M. 2013. Characterization of 532

extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of 533

Enterobacteriaceae causing cattle mastitis in France. Veterinary Microbiology 534

162:793-799. 535

44. Valverde A, Canton R, Garcillan-Barcia MP, Novais A, Galan JC, Alvarado A, 536

de la Cruz F, Baquero F, Coque TM. 2009. Spread of bla(CTX-M-14) is driven mainly 537

by IncK plasmids disseminated among Escherichia coli phylogroups A, B1, and D in 538

Spain. Antimicrobial Agents and Chemotherapy 53:5204-5212. 539

45. Oteo J, Cercenado E, Fernandez-Romero S, Saez D, Padilla B, Zamora E, Cuevas 540

O, Bautista V, Campos J. 2012. Extended-spectrum-beta-lactamase-producing 541

Escherichia coli as a cause of pediatric infections: report of a neonatal intensive care 542

unit outbreak due to a CTX-M-14-producing strain. Antimicrobial Agents and 543

Chemotherapy 56:54-58. 544


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

23

46. Cremet L, Caroff N, Giraudeau C, Dauvergne S, Lepelletier D, Reynaud A, 545

Corvec S. 2010. Occurrence of ST23 complex phylogroup A Escherichia coli isolates 546

producing extended-spectrum AmpC beta-lactamase in a French hospital. 547

Antimicrobial Agents and Chemotherapy 54:2216-2218.548


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

24

TABLE 1: Distribution and percentage of ESBL, AmpC and PMQR genes 549

Cze

ch R

epubli

c

Fra

nce

Ger

man

y

Ital

y

Pola

nd

,Gdynia

Pola

nd

,

Jaro

slaw

Ser

bia

Spai

n

Sw

itze

rlan

d

ESBL type 82 (100%)

CTX-M-1 17 - 8 - 13 1 - - - 39 (48 %)

CTX-M-8 1 - - - - - - - - 1 (1 %)

CTX-M-14 - - - - 1 - - 1 - 2 (2 %)

CTX-M-15 13 - 6 - 2 - 4 - - 25 (30 %)

CTX-M-24 3 - - - 1 - - - - 4 (5 %)

CTX-M-25 - - - - - 1 - - - 1 (1 %)

CTX-M-28 - - 1 - - - - - - 1 (1%)

CTX-M-55 - - 2 - - - - - - 2 (2 %)

TEM-52 3 - - - - 1 - - - 4 (5 %)

SHV-12 - - 2 - - - - - - 2 (2 %)

ND 1 - - - - - - - - 1 (1 %)

AmpC type

47 (100%)

CMY-2 27 - - - 18 2 - - - 47 (100 %)

PMQR type

62 (100%)

qnrS1 25 - - - 11 12 1 5 - 54 (87%)

qnrS2 1 - - - - - - - - 1 (2%)

qnrB19 - - - - 4 - - - - 4 (6%)

aac(6´)-Ib-cr - - - - - - 1 - - 1 (2%)

qnrS1+qnrB19 1 - - - 1 - - - - 2 (3%)

ND not detected550


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

25

TABLE 2: Distribution of ST types related to ESBL type in eight European countries 551

ES

BL

gen

e

ST

ST

C

Ph

ylo

gen

etic

gro

up

c

Cze

ch R

epu

bli

c

Ger

man

y

Po

lan

d,

Gd

yin

a

Po

lan

d,

Jaro

slaw

Ser

bia

Sp

ain

n

blaCTX-M-1 ST10a STC10 A 1 2 1

4

ST48a STC10 A 2

1

3

ST58a STC155 A×B1 7

7

ST101 STC101 B1

1

1

ST106 STC69 ABD

1

1

ST115 STC38 ABD

4

4

ST154 STC155 A×B1

1

1

ST206a STC10 A×B1 1 1

ST351a STC351 ABD

1

1

ST394 STC69 ABD

1

1

ST398a STC10 A×B1 1

1

ST542a STC542 ABD 1

1

ST609 STC10 A×B1

1

1

ST617a STC10 A 1

1

ST641a STC86 ABD

1

1

ST669 STC10 A 1

1

ST746a STC10 A 1

1

2

ST1011 STC1011 ABD

1

1

ST1141 STC10 A

1

1

ST1725 STC23 A×B1

1

1

ST1832 STC1832 ABD 2

2

ST2226 STC10 A

1

1

ST3017b STC155 A×B1 1 1

blaCTX-M-8 ST58a STC155 A×B1 1 1

blaCTX-M-14 ST1642 STC155 A×B1

1 1

ST3056b none ABD

1

1

blaCTX-M-15 ST10a STC10 A 1 1

ST131a STC131 B2 8

3

11

ST167a STC10 A

1 1

2

ST361a STC361 A×B1

1

1

ST398a STC10 A×B1

1

1

ST405 STC405 ABD 1

1

ST448a STC155 A×B1

1

1

ST617a STC10 A 4

4

ST1249 none A×B1

1

1

ST3015a,b

STC10 A

1

1

ST3018b none ABD

1

1

blaCTX-M-24 ST3014b none A 2 1 3

ST3020b STC10 A 1

1

blaCTX-M-25 ST155a STC155 A×B1 1 1


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

26



blaTEM-52 ST88 STC23 B1 1 1

ST1638 STC10 A 2

2

ST3019b none ABD 1

1

blaSHV-12 ST746a STC10 A 2 2

ND ST3223b STC131 B2 1 1

Prevalence

38

(25,3

%)

19 (19

%)

17

(11,3

%)

3 (2

%)

4 (2,7

%)

1 (0,7

%)

82 (7,6

%) aSTs capable to carrying different plasmid-mediated resistance genes (ESBL, AmpC, PMQR) in our

study; bnovel ST type;

cPhylogenetic groups were calculated using Structure programme, A×B1and ABD

are hybrid groups that were likely derived from their ancestry groups A and B1 (A×B1), or multiple

sources of ancestry (ABD); (7); ND not detected

552

553

554

555

556

557

558

559

560

561

562

563

564

565


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

27

TABLE 3: Distribution of ST types related to AmpC type in eight European countries 566

Am

pC

gen

e

ST

ST

C

Ph

ylo

gen

etic

gro

up

c

Cze

ch R

epu

bli

c

Po

lan

d,

Gd

yin

a

Po

lan

d,

Jaro

slaw

n

blaCMY-2 ST10a STC10 A 3

3

ST23a STC23 B1

1

1

ST57 STC57 ABD

1

1

ST58a STC155 A×B1 2 2

4

ST69a STC69 ABD 2

2

ST93a STC10 A 2

2

ST95 STC95 B2

1 1

ST117 STC117 ABD 1 2

3

ST131a STC131 B2

1 1

ST351a STC351 ABD 4

4

ST354 STC354 ABD 1

1

ST429 STC429 B2 3

3

ST453 STC86 ABD

3

3

ST615 STC10 AxB1

2

2

ST665 none ABD 1

1

ST770 STC770 D 1

1

ST963 STC38 D 3

3

ST1056 STC155 B1

1

1

ST1167 STC155 AxB1 2

2

ST1431 STC1431 B1

1

1

ST3274 STC10 A#

1

1

ST3568 none A#

1

1

ST3778 STC117 D# 1

1

ST4274b STC57 D

# 1

1

ST4275b STC10 A

# 1 1

ST4276b STC10 A

#

1

1

NT - -

1

1

Prevalence

27 (18

%)

18 (12

%)

2 (1,4

%)

47 (4,4

%) aSTs capable to carrying different plasmid-mediated resistance genes (ESBL, AmpC,

PMQR) in our study; bnovel ST type;

cPhylogenetic groups were calculated using

Structure programme, A×B1and ABD are hybrid groups that were likely derived from

their ancestry groups A and B1 (A×B1), or multiple sources of ancestry (ABD); (7);

NT non-typeable #Tested by multiplex PCR assay (26)

567

568

569


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

28

TABLE 4: Distribution of ST types related to PMQR type in eight European countries 570

PM

QR

gen

e

ST

ST

C

Ph

ylo

gen

etic

gro

up

c

Cze

ch R

epu

bli

c

Po

lan

d,

Gd

yin

a

Po

lan

d,

Jaro

slaw

Ser

bia

Sp

ain

n

qnrS1 ST10a STC10 A 1 1 1

3

ST23a STC23 B1

2

2

ST34 STC10 A 1

1

ST46 STC10 A

1

1

ST48a STC10 A 4

4

ST58a STC155 A×B1 2 1

3

ST69a STC69 ABD 1

1

2

ST93a STC10 A 1

1

ST155a STC155 A×B1 1 1

2

ST162 STC155 A×B1 1

1

ST206a STC10 A×B1

1

1

ST224 STC155 A×B1

1

1

ST345 STC23 B1

1

1

ST351a STC351 ABD 1

1

ST398a STC10 A×B1 1 1

2

ST399 STC399 A×B1 1

1

ST442 STC155 B1 1

1

ST450 STC4358 A 1

1

ST542a STC542 ABD

2

1 3

ST762 STC10 A

1 1

ST767 STC155 A×B1

1

1

ST1137 STC10 A 2

2

ST1251 STC10 A 1

1

ST1433 none A×B1

1

1

ST1582 STC155 A×B1

1

1

ST1720 ST86 ABD

1

1

ST1882 none ABD

1

1

ST2179 none A×B1 1

1

ST2526 none B1

1 1

ST2705 STC10 A

1

1

ST2722 STC155 B1

3

3

ST3270b STC10 A 3

3

ST3271b STC542 ABD

1 1

ST3273b STC10 A×B1

1

1

ST3309b STC4358 A 1

1

ST3310b STC23 B1 1 1

qnrS2 ST10a STC10 A 1 1

qnrB19 ST2491 STC10 A 2 2

ST3107 STC10 A×B1

1

1

ST3272b STC10 A 1 1

aac(6´)-Ib-cr ST90 STC23 A×B1 1 1


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

29

qnrS1+qnrB19 ST641a STC86 ABD 1

1

ST3272b STC10 A 1 1

Prevalence

27 (18

%)

16

(10,7

%)

12 (8,1

%)

2 (1,3

%)

5 (3,3

%)

62 (5,8

%) aSTs capable to carrying different plasmid-mediated resistance genes (ESBL, AmpC, PMQR) in

our study; bnovel ST type;

cPhylogenetic groups were calculated using Structure programme,

A×B1and ABD are hybrid groups that were likely derived from their ancestry groups A and B1

(A×B1), or multiple sources of ancestry (ABD); (7)

571

572

573

574

575

576

577

578

579

580

581

582

583

584


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

30

Figure legend: 585

FIGURE 1: eBURST diagram showing clustering of E. coli STs belonging to three main 586

complexes STC10, STC23 and STC155 that were isolated from feces of European rooks. 587

Each ST is represented as a node. Single locus variants are connected with a line. The STs 588

found in our study are displayed as numbered STs in a pink circle. Predicted founders are 589

shown in blue, the subgroup founders in yellow. Clonal complexes designations are based on 590

STs which originally constituted respective clonal complexes. Designations of complexes 591

constituted by merging of two or more former complexes are based on the designation of 592

oldest such (sub)complex. 593


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

ST10 (14)

ST23 (3) ST155 (3)

ST609 (1)

ST615 (2)

ST46 (1)

ST398 (4)

ST746 (4)

ST3273 (1)

ST3272 (2)

ST762 (1) ST2226 (1)

ST1638 (2)

ST3020 (1)

ST34 (1)

ST206 (2)

ST4275 (1)

ST167 (3)

ST617 (5)

ST3015 (1) ST48 (7) ST3270 (3)

ST669 (1)

ST93 (3)

ST1251 (1)

ST1137 (2)

ST1141 (1)

ST2705 (1)

ST2491 (2)

ST4276 (1)

ST3107 (1) ST3274 (1)

ST58 (15)

ST154 (1)

ST1056 (1)

ST442 (1)

ST224 (1) ST162 (1)

ST448 (1)

ST88 (1)

ST1167 (2)

ST767 (1)

ST1582 (1)

ST1642 (1)

ST2722 (3)

ST3017 (1)

ST90 (1) ST3310 (1)

ST345 (1)

ST1725 (1)


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

Plasmid-mediated resistance to cephalosporins and ...

Documents

Transcript of Plasmid-mediated resistance to cephalosporins and ...