First countrywide survey of third-generation cephalosporin-resistant Escherichia coli from broilers, swine, and cattle in Switzerland

Diagnostic Microbiology and Infectious Disease 73 (2012) 31–38

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Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o

First countrywide survey of third-generation cephalosporin-resistant Escherichia coli from broilers, swine, and cattle in Switzerland☆ Andrea Endimiani1, Alexandra Rossano, Daniel Kunz, Gudrun Overesch, Vincent Perreten ⁎ Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland

a r t i c l e

i n f o

Article history: Received 4 November 2011 Received in revised form 9 January 2012 Accepted 10 January 2012 Keywords: ESBL CTX-M AmpC CMY Animal

a b s t r a c t The herd prevalence of third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) was determined for broilers (25.0% [95% confidence interval (CI) 17.6–33.7%]), pigs (3.3% [(95% CI 0.4–11.5%]), and cattle (3.9% [95% CI 0.5–13.5%]), using a sampling strategy that was representative of the livestock population slaughtered in Switzerland between October 2010 and April 2011. The 3GC-R-Ec isolates were characterized by the measurement of the MICs of various antibiotics, microarray analyses, analytical isoelectric focusing, polymerase chain reaction and DNA sequencing for bla genes, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. CMY-2 (n = 12), CTX-M-1 (n = 11), SHV-12 (n = 5), TEM-52 (n = 3), CTX-M-15 (n = 2), and CTX-M-3 (n = 1) producers were found. The majority of CMY-2 producers fell into 1 PFGE cluster, which predominantly contained ST61, whereas the CTX-M types were carried by heterogeneous clones of E. coli, as shown by the numerous PFGE profiles and STs that were found. This is the first national Swiss study that focuses on the spread of 3GC-R Enterobacteriaceae among slaughtered animals. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Over the past 2 decades, there has been an increasing number of infections worldwide due to third-generation cephalosporin-resistant (3GCs-R) Escherichia coli isolates (Coque et al., 2008; Hawser et al., 2011; Rosenthal et al., 2010). The production of Ambler class A extended-spectrum β-lactamases (ESBLs) and class C plasmidmediated AmpC (pAmpC) enzymes is the most encountered mechanism responsible for this phenomenon. TEM, SHV, and CTX-M types are the 3 main families of ESBLs, whereas 6 families of pAmpCs (CMY, FOX, DHA, MOX, ACC, ACT types) have been described (Jacoby, 2009; Perez et al., 2007). To date, the most frequently detected ESBLs in E. coli are of the CTX-M types (Oteo et al., 2010; Peirano and Pitout, 2010), whereas CMY-2 is the most recurrent pAmpC (Jacoby, 2009). E. coli is the major pathogen responsible for urinary tract and bloodstream infections in humans (Rosenthal et al., 2010). Some pathogenic E. coli isolates are also frequently responsible for diarrheal infections, and livestock plays an important role as reservoir (Kaper et al., 2004). Currently, the presence of 3GCs-R E. coli (3GCs-R-Ec) in clinical settings represents a public-health concern because these infections are challenging the therapeutic armamentarium (Giamarellou and Poulakou,

☆ The Swiss Federal Veterinary Office and the Canton of Bern financed this study. ⁎ Corresponding author. Tel.: +41-31-631-2430; fax: +41-31-631-2634. E-mail address: [email protected] (V. Perreten). 1 Present address: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, Postfach 61, CH-3010, Switzerland. Tel.: +41-31-632-8632; fax: +41-31632-8766. 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2012.01.004

2009; Pitout, 2010). In fact, ESBL and pAmpC genes are usually carried on mobile plasmids along with other gene-resistance traits (e.g., those for quinolones and aminoglycosides) that render the isolates multidrug resistant (Jacoby, 2009; Perez et al., 2007). As a result, one important task is to monitor the prevalence of these genetic elements among E. coli in community, hospital, and environmental settings to implement new strategies that would limit the spread of these elements to lifethreatening pathogens. Healthy animals can be important reservoirs of Gram-negative species that carry genes conferring resistance to β-lactams and other antimicrobial classes. Currently, CTX-M–type ESBLs and CMY-2 pAmpC are increasingly reported in numerous countries, mainly among strains of E. coli and Salmonella spp. that colonize food-producing animals (e.g., cattle, pigs) and animal companions (Carattoli, 2008; European Food Safety Authority Panel on Biological Hazards (BIOHAZ), 2011; Li et al., 2007). However, in poultry, CMY-2–positive E. coli isolates are more rarely described and usually have a lower prevalence than the ESBL producers have (Blanc et al., 2006; Dierikx et al., 2010; European Food Safety Authority Panel on Biological Hazards (BIOHAZ), 2011; Leverstein-van Hall et al., 2011; Li et al., 2007, 2010). Only Smet et al. (2008) reported a high prevalence (i.e., 49%) of CMY-2–positive E. coli isolates in Belgian broiler farms. In Switzerland, national phenotypic surveillances from the past 2 years indicate that the prevalence of 3GCs-R Enterobacteriaceae in humans and in food-producing animals is lower than in other European countries (Büttner et al., 2010, 2011; European Food Safety Authority Panel on Biological Hazards (BIOHAZ), 2011) (http://www. search.ifik.unibe.ch/en/index.html). Nevertheless, data regarding the

2 2 1 1

1 1 1

3/11 (27.3) 2

5/22 (22.7) 1 2 1 1

0/1 0/2 2/20 (10.0) 2 1/1

AG = Aargau; BE = Bern; FR = Fribourg; GE = Geneva; JU = Jura; LU = Lucerne; SG = St. Gallen; SH = Schaffhausen; SO = Solothurn; TG = Thurgau; VD = Vaud; VS = Valais; ZG = Zug; ZH = Zurich.

1

1/1 (100) 0/2 3/9 (33.3)

6/20 (30.0) 4 1 1/1 (100) 1/1 1/1 (100)

0/3

0/1 0/1

0/3

2/7 (28.6) 1 1

2/4

0/1 0/1 6/18 (33.3) 2 2

0/1

0/1

0/1

1/1 0/1

0/1

ZG (1)

0/1

VD (17) TG (9)

1/2 5/14 1/2 0/3 1/2

SO (7) SH (3)

0/2

SG (3)

0/2

LU (20)

6/13 0/3 0/4

JU (1)

1/1 1/7 3/14

GE (1) FR (20)

0/3 2/14

BE (22) AG (11)

2/4 0/1 1/6

A B C D E All slaughterhouses (%) CMY-2 CTX-M-1 CTX-M-15 SHV-12 TEM-52

No. of 3GCs-R-Ec isolates/overall no. of different holdings (n) in the different Swiss Cantons

Slaughterhouse and 3GCs-R groups

Table 1 Epidemiologic data regarding third-generation cephalosporin-resistant E. coli (3GCs-R-Ec) isolates from broilers in Switzerland.

1/4 0/1

VS (1)

ZH (2)

Unknown (1)

13/43 (30.2) 11/51 (21.6) 2/12 (16.7) 3/10 (30.0) 1/4 (25.0) 30/120 (25.0) 12/30 (40.0) 9/30 (30.0) 1/30 (3.3) 5/30 (16.7) 3/30 (10.0)

A. Endimiani et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 31–38 All holdings (%)

32

molecular mechanisms responsible for resistance to 3GCs are still lacking. In a recent pilot study at only 1 slaughterhouse, feces from swine and cattle that were sampled in October 2009 were analyzed to determine the percentage of ESBL-producing Enterobacteriaceae. The results demonstrated that 15.2% of the pigs and 17.1% of the cattle were positive, and the CTX-Ms were the only ESBLs found (Geser et al., 2011). However, the possible presence of pAmpCs was not taken into account, and neither the type of blaCTX-M and blaTEM nor the resistance genes against other antibiotics were determined (Geser et al., 2011). Additionally, no data are yet available about the distribution and the molecular characteristics of 3GC-R-Ec in broiler production in Switzerland. In the present study, we used a sampling strategy evenly distributed throughout the months and years and representative for the contemporary Swiss livestock population. We then implemented standard molecular and biochemical tests to detect all possible ESBL and pAmpC producers among the 3GC-R-Ec isolates from broilers, cattle, and swine. 2. Materials and methods 2.1. Sample collection Representative samples were taken according to the guidelines of the Swiss National Monitoring Program on Antimicrobial Resistance in Food Animals (Büttner et al., 2011). The sampling strategy consists of collecting from each slaughterhouse a number of samples that are proportional to the number of animals slaughtered at each establishment per year. The sampling is also evenly distributed across each month of the study period. The samples were randomly collected at the 5 biggest broiler abattoirs (5 to 207 samples per slaughterhouse per year) and at the 9 biggest pig (10 to 102 samples) and 7 biggest cattle (2 to 66 samples) abattoirs where over 80% of livestock in Switzerland are slaughtered. Only 1 sample was taken per animal holding for pigs and cattle, and 1 pool of 5 animals per holding was analyzed for broilers. For this study, broiler samples taken from October 18, 2010, through April 30, 2011, and pig and cattle samples taken from January 1, 2011, through April 30, 2011, were analyzed for the presence of 3GC-R isolates. 2.2. Detection of third-generation cephalosporin-resistant isolates Five broiler cloacal swabs per holding were vortexed together for 30 s in 1 mL of Tryptone Soy Broth (Becton Dickinson, Franklin Lakes, NJ). This suspension and the fecal swabs from pigs and cattle were transferred into 5 mL of MacConkey broth (Oxoid, Basingstoke, UK) containing ceftazidime (8 mg/L) and incubated at 37 °C for 24 h under agitation. Then, 1 full loop (10 µL) was plated onto selective chromogenic medium for the screening of 3GCs-R Enterobacteriaceae (chromID ESBL; bioMérieux, Marcy l'Etoile, France) and reincubated overnight. From each selective plate, a single colony from those showing a unique color and morphology as described in the manufacturer's product documentation (bioMérieux) was further identified to the species level. 2.3. Species identification and antimicrobial susceptibility tests Species identification (ID) and antimicrobial susceptibility tests (ASTs) were routinely assessed using the Vitek 2 system on AST-GN38 cards (bioMérieux). The ID was confirmed with matrix-assisted laser desorption/ionization time of flight mass spectrometry (microflex LT, Bruker Daltonik, Bremen, Germany). The MICs were determined by microdilution in Mueller-Hinton broth (BBL, Becton Dickinson) using the Sensititre ESB1F plate (Trek Diagnostics Systems, East Grinstead, England) according to the Clinical and Laboratory Standard Institute (CLSI) guidelines (Clinical and Laboratory Standards Institute, 2009).

Table 2 Epidemiologic data regarding 3GC-R E. coli isolates from swine (S) and cattle (C) in Switzerland. No. of 3GC-R E. coli isolates/overall no. of different husbandry (n) in the different Swiss Cantons AG

S (6) F G H I J K L M N O P Q All slaughterhouses CTX-M-1 CTX-M-3 CTX-M-15

AI

C (1)

S (4)

BE

C S (6)

FR

C (16)

S (4)

0/5 0/8

C (5)

GE

GR

JU

LU

S C (1)

S C (2)

S C (3)

S (8)

0/1

0/1

0/4 0/1

0/1 0/1 1/1

OW

C (5)

S C (1)

S (1)

0/2 0/1 0/1

0/1

SG

C S (15)

0/3

0/1

0/2 0/1

C (1)

SH

SO

S C (2)

S (1)

C (1)

SZ

TG

S C (1)

S (9)

0/2

0/1

1/6

0/1

0/4

-

0/6

0/16

0/1 0/4

0/1

0/4 0/1

0/1

0/1 0/5 0/1

0/2

0/5

0/1

-

0/2

0/3

0/8

C (1)

S (1)

ZH

C (2)

S (3)

0/1

C (4)

U

All holdings (%)

C (1)

S (60)

C (51)

0/8 2/5 0/4 0/2 0/16 0/12 0/9 0/2 0/2 2/60 3.3% 1/60 1/60 -

2/16 0/12 0/10 0/4 0/3 0/5 0/1 2/51 3.9% 1/51 1/51

1/1 0/2

0/1

0/1 0/1

0/1

1/4

0/1

0/1

0/4 0/3

S (2)

0/1

0/1

0/6 1/2

0/3

0/1

C (5)

ZG

0/1

0/1 0/5

VD

1/1

0/1

0/1 0/3

NE

0/5

-

0/1

0/1

-

1/15

0/1

-

1/2

1

0/1

0/1

-

0/1

0/1 0/3 0/2 0/1 0/2 0/9

0/1 0/1 0/1 0/1

0/5

0/1 0/2

0/1

0/1

0/2

0/3

1

1 1

AG = Aargau; AI = Appenzell Innerrhoden; BE = Bern; FR = Fribourg; GE = Geneva; GR = Graubünden; JU = Jura; LU = Lucerne; NE = Neuchâtel; OW = Obwald; SG = St. Gallen; SH = Schaffhausen; SO = Solothurn; SZ = Schwytz; TG = Thurgau; VD = Vaud; ZG = Zug; ZH = Zurich; U = unknown origin.

A. Endimiani et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 31–38

Slaughterhouse and 3GCs-R groups

33

34

Table 3 Antimicrobial susceptibility test results and molecular characteristics of the thirty-four 3GC-R-Ec isolates collected from food-producing animals in Switzerland. Isolate

Phenotypic data (MIC, mg/L)a

Biochemical and molecular characterizations FEP

IPM

MEM

CIP

GEN

TOB

AK

SXT

TET

NIT

PolB

aIEF

Check-Points microarray CT-101 and amino acid substitutionsb

PCR/DNA sequencing for bla genes

Identibac microarray (AMR-ve)c

16 16

16 16

≤0.5 ≤0.5

≤0.25 ≤0.25

≤0.5 ≤0.5

≤0.5 ≤0.5

≤2 ≤2

≤1 ≤1

4 ≤2

≤20 ≥320

2 ≥16

64 ≤16

1 0.5

9.0 5.4, 9.0

CMY-II CMY-II, TEM (all)

CMY-2 CMY-2, TEM-1

16 32 32 64 4

32 16 64 64 8

32 16 32 32 4

≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25

≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

≤2 ≤2 ≤2 ≤2 ≤2

≤1 ≤1 ≤1 ≤1 ≤1

≤2 ≤2 4 ≤2 ≤2

≤20 ≤20 ≤20 ≤20 40

≤1 ≤1 ≤1 ≤1 ≥16

≤16 ≤16 ≤16 ≤16 ≤16

1 0.5 1 1 0.5

9.0 9.0 5.6, 9.0 9.0 5.4, 9.0

CMY-II CMY-II CMY-II CMY-II CMY-II, TEM (all)

CMY-2 CMY-2 CMY-2 CMY-2 CMY-2, TEM-1

64 64 32 64 128

32 32 32 64 64

32 ≥64 16 32 ≥64

32 32 16 32 32

≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25

≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

≤2 ≤2 ≤2 ≤2 ≤2

≤1 ≤1 ≤1 ≤1 ≤1

≤2 ≤2 ≤2 ≤2 ≤2

≤20 ≤20 ≤20 ≤20 ≥320

≤1 ≤1 ≤1 ≤1 2

≤16 ≤16 ≤16 ≤16 64

0.5 0.5 1 0.5 0.5

9.0 9.0 9.0 9.0 9.0

CMY-II CMY-II CMY-II CMY-II CMY-II

CMY-2 CMY-2 CMY-2 CMY-2 CMY-2

CMY(all) CMY(all), TEM(all), int1, aadA1, sul1, dfrA1, tet(A) CMY(all), CMY(all) CMY(all), CMY(all) CMY(all), TEM(all), aadA1, dfrA5, tet(A), strB CMY(all) CMY(all), CMY(all) CMY(all), CMY(all), int2, aadA1, dfrA1, sul2

16 64 ≤2

64 128 1

32 64 ≤0.06

32 ≥64 ≥64

32 32 ≤0.06

≤0.5 ≤0.5 8

≤0.25 ≤0.25 ≤0.25

≤0.5 ≤0.5 ≤0.5

≤0.5 ≤0.5 ≤0.5

≤2 ≤2 ≤2

≤1 ≤1 ≤1

≤2 4 ≤2

≤20 ≥320 ≤20

≤1 ≥16 ≥16

≤16 64 64

0.5 1 0.5

8.4, 9.2

CTX-M-I

CTX-M-1

≤2

≤2

2

≤0.06

32

≤0.06

8

≤0.25

≤0.5

≥4

≤2

≤1

4

≥320

≥16

≤16

1

5.4, 8.4

CTX-M-I, TEM (all)

CTX-M-1, TEM-1

Msa971

≤2

≤2

≤0.12

≤0.06

32

≤0.06

4

≤0.25

≤0.5

≤0.5

≤2

≤1

4

≥320

≥16

64

0.5

5.4, 8.4, 9.2

CTX-M-I, TEM (all)

CTX-M-1, TEM-1

Msa994

≤2

≤2

≤0.12

≤0.06

16

≤0.06

2

≤0.25

≤0.5

≤0.5

≤2

≤1

≤2

≥320

≥16

≤16

0.5

8.6

CTX-M-I

CTX-M-1

Msa1005 11/IMD0035

≤2 4

≤2 ≤2

0.5 2

≤0.06 ≤0.06

32 32

≤0.06 ≤0.06

2 8

≤0.25 ≤0.25

≤0.5 ≤0.5

≤0.5 ≤0.5

≤2 ≤2

≤1 8

≤2 ≤2

≤20 ≥320

≥16 ≥16

64 64

0.5 0.5

8.4, 8.8 5.4, 8.4

CTX-M-I CTX-M-I, TEM (all)

CTX-M-1 CTX-M-1, TEM-1d

11/IMD0040 11/IMD0041

4 4

≤2 ≤2

1 8

≤0.06 0.25

≥64 ≥64

≤0.06 ≤0.06

4 4

≤0.25 ≤0.25

≤0.5 ≤0.5

≤0.5 ≤0.5

≤2 ≤2

≤1 ≤1

≤2 ≤2

≤20 ≥320

≥16 ≥16

≤16 32

0.5 0.5

5.6, 8.4 8.6, 8.8

CTX-M-I CTX-M-I

CTX-M-15 CTX-M-1

11/IMD0042 11/IMD0077

4 8

≤2 ≤2

1 8

0.25 ≤0.06

32 ≥64

≤0.06 ≤0.06

4 4

≤0.25 ≤0.25

≤0.5 ≤0.5

≤0.5 ≤0.5

≤2 ≤2

≤1 ≤1

≤2 ≤2

≤20 40

≥16 2

≤16 ≤16

0.5 0.5

8.4 8.0, 9.0

CTX-M-I CTX-M-I

CTX-M-1 CTX-M-15

11/IMD0081

≤2

≤2

16

≤0.06

≥64

≤0.06

≤0.5

≤0.25

≤0.5

≤0.5

≤2

8

≤2

≤20

≤1

≤16

0.5

5.4, 8.8, 8.9

CTX-M-I, TEM (all)

CTX-M-3d, TEM-1

11/IMD0190

≤2

≤2

1

≤0.06

16

≤0.06

2

≤0.25

≤0.5

≤0.5

≤2

≤1

4

≥320

≥16

64

0.5

8.8

CTX-M-I

CTX-M-1

11/IMD0204 11/IMD0513

≤2 8

≤2 ≤2

0.5 1

≤0.06 ≤0.06

32 32

≤0.06 ≤0.06

4 ≤0.5

≤0.25 ≤0.25

≤0.5 ≤0.5

≤0.5 ≤0.5

≤2 ≤2

≤1 ≤1

≤2 4

≥320 ≥320

≥16 ≥16

≤16 64

0.5 0.5

8.4 8.4

CTX-M-I CTX-M-I

CTX-M-1 CTX-M-1

MIC50 MIC90 Msa899

≤2 8 8

≤2 ≤2 ≤2

1 8 8

≤0.06 0.25 ≤0.06

32 ≥64 2

≤0.06 ≤0.06 ≤0.06

4 8 ≤0.5

≤0.25 ≤0.25 ≤0.25

≤0.5 ≤0.5 ≤0.5

≤0.5 ≤0.5 ≤0.5

≤2 ≤2 ≤2

≤1 8 ≤1

≤2 4 ≤2

≥320 ≥320 ≤20

≥16 ≥16 ≥16

16 64 ≤16

0.5 0.5 1

8.2, 8.6, 8.8

SHV: G238S, E240K

SHV-12

Msa900

≤2

≤2

8

≤0.06

2

≤0.06

≤0.5

≤0.25

≤0.5

≤0.5

≤2

≤1

≤2

≤20

≥16

≤16

1

8.2, 8.6, 8.8

SHV: G238S, E240K

SHV-12

Msa902

≤2

≤2

16

≤0.06

4

≤0.06

≤0.5

≤0.25

≤0.5

≤0.5

≤2

≤1

4

≤20

≥16

≤16

0.5

8.2

SHV: G238S, E240K

SHV-12

TZP

CAZ

CAZ CLA

Msa893 Msa901

≥128 ≥128

≤2 8

32 32

16 16

Msa970 Msa972 Msa991 Msa992 Msa1088

≥128 ≥128 ≥128 ≥128 64

8 8 32 16 ≤2

64 32 64 64 8

11/IMD0062 11/IMD0087 11/IMD0129 11/IMD0147 11/IMD0209

≥128 ≥128 ≥128 64 ≥128

16 16 8 32 64

MIC50 MIC90 Msa936

≥128 ≥128 ≤2

Msa937

CTX

CTX-M-1, sul2, tet(A), strA/B CTX-M-1, TEM(all), int1, aadA1, dfrA1, catA1, tet(A), sul2, strB CTX-M-1, TEM(all), int1, aadA1/2, dfrA1, sul2, tet(A), catA1 CTX-M-1, int1, aadA4, dfrA17/19, sul2, tet(B), strB CTX-M-1, sul2, tet(A) CTX-M-1, TEM(all), int1, catA1, strB, aadA1/4, dfrA1/17, sul1/2, tet(B) CTX-M-1, sul2, tet(A) CTX-M-1, sul2, tet(A), dfrA17/19 CTX-M-1, sul2, tet(A) CTX-M-1, int2, aadA1, dfrA1 CTX-M-1, TEM(all), int1, aadA1, sul1/2, strA/B CTX-M-1, int1, aadA4, dfrA17/19, sul2, tet(A) CTX-M-1, sul2, tet(A) CTX-M-1, int1, aadA1, dfrA1/17, sul2, tet(A)

SHV(all), int1, aadA1/2, sul3, tet(A), cmlA1 SHV(all), int1, aadA1/2, sul3, tet(A), cmlA1 SHV(all), int1, aadA1/2, sul2/3, tet(A), cmlA1

A. Endimiani et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 31–38

CTX CLA

FOX

FOX = Cefoxitin; TZP = piperacillin–tazobactam; CAZ = ceftazidime; CTX = cefotaxime; FEP = cefepime; IPM = imipenem; MEM = meropenem; CLA = clavulanate (constant concentration of 4 mg/L); CIP = ciprofloxacin; GEN = gentamicin; TOB = tobramycin; AK = amikacin; SXT = trimethoprim/sulfamethoxazole; TET = tetracycline; NIT = nitrofurantoin; PolB = polymyxin B. Antibiotic resistance genes and integrases: aadA1, aadA2, and aadA4 = aminoglycoside nucleotidyltransferase gene for streptomycin and spectinomycin resistance; catA1 = chloramphenicol acetyltransferase gene; cmlA1 = chloramphenicol efflux gene; dfrA1, dfrA5, dfrA17, and drfA19 = dihydrofolate reductase gene for trimethoprim resistance; tet(A) and tet(B) = tetracycline efflux gene; strA and strB = streptomycin phosphotransferase gene; sul1, sul2, and sul3 = dihydropteroate synthase gene for sulfonamide resistance; int1 and int2 = class I and II integrases. a MICs for TOB, AK, SXT, TET, NIT, and PolB were obtained using the Vitek system, whereas all of the others using the Sensititre ESB1F plate (Trek Diagnostics Systems). b TEM (all) indicates that only broad-spectrum TEM type (e.g., TEM-1) were found. The amino acid substitutions for SHV- and TEM-type ESBL are indicated. c CMY (all), TEM (all), and SHV (all) indicate that the microarray is not able to distinguish subclasses of the β-lactamases (e.g., SHV- and TEM-type broad-spectrum enzymes from those with ESBL spectrum). d Double spikes in the DNA sequence were observed.

TEM-52d TEM-52d TEM: E104K, G238S TEM: E104K, R164H, G238S ≤16 ≤16 ≤16 64 ≤1 ≤1 ≥16 ≥16 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤2 ≤2 ≤2 ≤2 ≤2 8 ≤2 8 11/IMD0050 11/IMD0063 MIC50 MIC90

8 8 8 16

≤0.06 ≤0.06 ≤0.06 ≤0.06

4 8 2 8

≤0.06 ≤0.06 ≤0.06 ≤0.06

≤0.5 ≤0.5 ≤0.5 ≤0.5

≤0.5 ≤0.5 ≤0.5 ≤0.5

≤2 ≤2 ≤2 ≤2

≤1 ≤1 ≤1 ≤1

≤2 ≤2 ≤2 4

≤20 ≤20 ≤20 ≥320

1 0.5 0.5 1

5.6 5.6

TEM-52d TEM: R164H; SHV: G238S, E240K ≤16 ≥16 ≤0.5 ≤0.25 ≤2 ≤2 11/IMD0477

8

≤0.06

2

≤0.06

≤0.5

≤0.5

≤2

≤1

≤2

≤20

0.5

5.4

SHV-12, TEM-1 SHV: G238S, E240K; TEM (all) 64 ≥16 ≤0.5 ≤0.25 ≤2 ≤2 11/IMD0191

16

≤0.06

8

≤0.06

≤0.5

≤0.5

≤2

≤1

≤2

≥320

0.5

5.4, 8.2

SHV-12 ≤16 ≥16 ≤0.5 ≤0.25 ≤2 ≤2 Msa1089

8

≤0.06

2

≤0.06

≤0.5

≤0.5

≤2

≤1

≤2

≤20

0.5

8.2

SHV: G238S, E240K

SHV(all), aadA1/2, sul3, tet(A), cmlA1 SHV(all), TEM(all), int1, aadA1/2, dfrA1, sul1, tet(A) SHV(all), TEM(all), int1, aadA1/2, sul3, tet(A), cmlA1 TEM(all) TEM(all)

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35

Susceptibility results were interpreted according to the current CLSI criteria (Clinical and Laboratory Standards Institute, 2011). 2.4. Microarray analyses Genomic extraction was performed using the DNA Bacteria Plus kit (QIAGEN, Hilden, Germany). The Check-Points microarray platform CT-101 (Check-Points, Wageningen, The Netherlands) was used as previously described to screen the 3GCs-R-Ec isolates for the presence of class A (blaTEM, blaSHV, blaCTX-M, blaKPC), pAmpC (blaCMY, blaFOX, blaDHA, blaMOX, blaACC, blaACT), and class B (blaNDM) β-lactamase genes (Endimiani et al., 2010b; Bogaerts et al., 2011). The Identibac microarray AMR-ve version 05m (Alere, Urdorf, Switzerland) was also used to characterize the 3GC-R-Ec isolates. The platform can identify genes conferring resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, sulfonamides, trimethoprim, phenicols, and class I and II integron-associated integrases (Batchelor et al., 2008). 2.5. Identification and classification of β-lactamase genes Polymerase chain reaction and DNA sequence analyses for class A (blaTEM, blaSHV and blaCTX-M) and pAmpC (blaCMY, blaFOX, blaDHA, blaMOX, blaLAT, blaACC, blaMIR) determinants were performed for all 3GC-R isolates using the primers and conditions that were previously reported (Perez-Perez and Hanson, 2002; Endimiani et al., 2009). DNA traces were analyzed using the Sequencher 4.10.1 software (Gene Codes Corporation, Ann Arbor, MI). Amino acid sequences were obtained using the ExPASy Proteomics Server (http://ca.expasy.org). The final β-lactamase proteins were compared to those that were previously described (http://www.lahey.org/studies/). 2.6. Analytical isoelectric focusing β-Lactamase preparations for analytical isoelectric focusing (aIEF) were obtained as previously described (Endimiani et al., 2009). Electrophoresis was performed on gels with a pH range of 3.5 to 10 (GelCompany, San Francisco, CA), using a Multiphor II apparatus (Amersham Biosciences, Uppsala, Sweden). The β-lactamases were detected by the addition of 1 mmol/L nitrocefin (Becton Dickinson Biosciences). One well-characterized Klebsiella pneumoniae isolate (VA367) that produced the TEM-1 (pI, 5.4), KPC-2 (pI, 6.7), SHV-11 (pI, 7.6), and SHV-12 (pI, 8.2) β-lactamases and 1 E. coli strain (9217/10) that expressed the CMY-2 (pI, 9.0) β-lactamase were used as controls (Rice et al., 2008; Endimiani et al., 2011). 2.7. Analysis of clonality The genetic relatedness of the 3GCs-R-Ec isolates was determined by pulsed-field gel electrophoresis (PFGE) using XbaI digests, as previously reported (Liesegang and Tschäpe, 2002). The PFGE was run at 14 °C on a CHEF III apparatus (Bio-Rad, Hercules, CA) for 20 h at 6 V/cm with a pulse-time ramping from 2 to 64 s. The band patterns were interpreted using the BioNumerics software version 5.1 (Applied Maths, Sint-Martens-Latem, Belgium) and applying the Pearson correlation similarity coefficient (tolerance of 1% and optimization of 0.5%); a phylogenetic tree was constructed using the unweighted pair-group method with arithmetic mean. The ST of all 3GC-R-Ec isolates was also obtained implementing the multilocus sequence typing (MLST) Pasteur's scheme (http://www.pasteur.fr/recherche/ genopole/PF8/mlst/). 2.8. Statistical analysis Sampling calculations were performed using the Win Episcope 2.0 software (http://www.clive.ed.ac.uk), as previously described

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A total of 600 cloacal samples representing 120 different broiler herds from 14 Swiss cantons were collected from 5 different slaughterhouses (A–E) during a 6-month period (i.e., October 18, 2010, through April 30, 2011) (Table 1). The fecal samples from pigs (n = 60) and from cattle (n = 51) were collected during a 4-month period from 9 (I–Q) and 7 (F–L) slaughterhouses, respectively (January 1, 2011, to April 30, 2011). The 60 pig samples originated from 60 different herds from 12 different cantons, and the 51 cattle samples originated from 51 different herds from 16 different cantons (Table 2). Even if the samples were taken during the coldest seasons of the year, these periods allowed to give a representative overview of the situation of 3GCs-R-Ec in animals in Switzerland, since seasonal variations in the prevalence of intestinal E. coli carriage were never observed during the National Antimicrobial Monitoring of Resistance for enteric bacteria (Büttner et al., 2010, 2011). Overall, 34 samples were positive for at least 1 3GCs-R-Ec isolate (other 3GCs-R species of Enterobacteriaceae were not detected). The overall prevalences of 3GCs-R-Ec isolates among the broiler, swine, and cattle herds were 25.0% (95% CI 17.6–33.7%), 3.3% (95% CI 0.4–11.5%), and 3.9% (95% CI 0.5–13.5%), respectively. In the broiler production, 3GCs-R-Ec isolates were detected in holdings from 9 different cantons and at each slaughterhouse (Table 1). In contrast, 3GCs-R-Ec isolates from swine and cattle were only detected in animals slaughtered at 2 of the slaughterhouses (Table 2). Recently, the European Food Safety Authority reported the frequencies of isolation of 3GCs-R-Ec in 2007, 2008, and 2009 in food-producing animals. In particular, the prevalences of 8.5% (range 1–26.4%), 2.3% (range 0–3.8%), and 1.6% (range 0–6.5%) were observed in the participating European countries for poultry, swine, and cattle, respectively, in 2009 (European Food Safety Authority Panel on Biological Hazards (BIOHAZ), 2011). The percentage of 3GCsR-Ec in Swiss broilers (25.0%), pigs (3.3%), and cattle (3.9%) is within the range of that observed in other European countries.

methods may have contributed to the possible underestimation of the CMY types and other pAmpCs among Enterobacteriaceae (Doi and Paterson, 2007; Munier et al., 2010). We therefore used standard molecular and biochemical tests, such as the aIEF, to detect all possible pAmpC producers. The second group of 3GCs-R-Ec isolates (n = 14; 41.2%) likely included CTX-M–type producers. In fact, cefoxitin and piperacillin/ tazobactam were in the susceptible ranges, cefotaxime was highly resistant (MIC90 ≥64 mg/L), ceftazidime and cefepime showed moderately increased MICs (both with MIC90 of 8 mg/L), and clavulanate recovered the activity of ceftazidime and cefotaxime (MIC90 of 0.25 and ≤0.06 mg/L, respectively). According to the CheckPoints microarray, all of these strains were carrying group I CTX-M β-lactamase gene(s). This finding was consistent with the production of β-lactamases with pI values of approx. 8.4–9.2. DNA sequencing identified CTX-M-1 as the most common ESBL (Table 3). The CTX-M-1–like (e.g., CTX-M-1, -15, and -32) ESBLs are widely distributed among E. coli isolates of animal and human origin (Carattoli, 2008; Coque et al., 2008; Perez et al., 2007). In particular, CTX-M-1 is first in rank in poultry from the Netherlands (Leversteinvan Hall et al., 2011), France (Girlich et al., 2007), and Belgium (Smet et al., 2008); in pigs from Spain (Cortés et al., 2010) and Portugal (Gonçalves et al., 2010); and in animal companions in the Czech Republic, Italy, and Portugal (Carattoli, 2008; Dolejska et al., 2011). The third group of 3GCs-R-Ec isolates (n = 8; 23.5%) showed phenotypic characteristics that were similar to those of the second group (i.e., CTX-M-1 producers). However, cefotaxime had lower MIC values (MIC90 of 8 mg/L), and the isolates were consistently fully susceptible to cefepime (MIC90 ≤0.5 mg/L). Five SHV-12– and 3 TEM52–producing isolates were identified by DNA sequencing. CheckPoints microarrays and aIEF results were consistent with these findings (Table 3). SHV-12– and TEM-52–producing E. coli and Salmonella isolates in food-producing animals and pets have been frequently described in different countries (Carattoli, 2008; Coque et al., 2008; European Food Safety Authority Panel on Biological Hazards (BIOHAZ), 2011). It should also be noted that SHV-12 and TEM-52 are among the most frequently detected ESBLs in Enterobacteriaceae responsible for infection in humans (Bush and Fisher, 2011; Coque et al., 2008; Endimiani et al., 2004).

3.2. Mechanisms of resistance against 3GCs

3.3. Other antibiotic resistance genes

Three different groups of 3GCs-R-Ec isolates could be distinguished according to the MIC values recorded for the β-lactams and β-lactam/ β-lactamase inhibitor combinations (Table 3). The first group of 3GCs-R-Ec isolates (n = 12; 35.3%) showed phenotypes that were suspicious for the production of pAmpCs. In particular, cefoxitin, piperacillin/tazobactam, ceftazidime, and cefotaxime were within the resistant range (MIC90 ≥64 mg/L); clavulanate was unable to recover the activity of ceftazidime and cefotaxime (MIC90 of 64 and 32 mg/L, respectively), whereas cefepime showed very potent in vitro activity (MIC90 of ≤0.5 mg/L) (Table 3). The microarray analysis using the Check-Points system indicated that all of these E. coli isolates were carrying CMY-2–like pAmpC β-lactamase(s). This finding was supported by the observation that all strains produced β-lactamase(s) with a pI of approx. 9.0, which is consistent with the production of CMYtype enzymes (Endimiani et al., 2010a). DNA sequencing of the amplified blaCMY gene confirmed the production of the CMY-2 pAmpC in all of the 3GCs-R-Ec isolates examined (Table 3). All of the CMY-2–producing E. coli isolates were found in poultry (Table 1), unlike in other studies that mainly reported ESBL producers (Blanc et al., 2006; Dierikx et al., 2010; Girlich et al., 2007; Leversteinvan Hall et al., 2011; Li et al., 2007, 2010). In this context, we should consider that the lack of simple and reliable phenotypic tests for screening and the limited implementation of specific molecular

As shown in Table 3, none of the 3GCs-R-Ec isolates carried plasmid-mediated, quinolone-resistance (PMQR) determinants; only 1 was resistant to ciprofloxacin, likely due to amino acid(s) substitutions in GyrA and/or ParC. This relative absence of isolates with reduced susceptibility to quinolones was unusual compared with the human clinical isolates. In Europe, resistance rates of 71–88% have been observed for ciprofloxacin among ESBL- or pAmpC-producing E. coli isolates of human origin (Hawser et al., 2010). Furthermore, although data regarding the presence of PMQR determinants in 3GCsR-Ec isolates of animal origin are still scarce, prevalence values of 17–35% have been recorded (Dolejska et al., 2011; Ma et al., 2009). Different degrees of resistance among the 3 groups of 3GCs-R-Ec isolates were observed for trimethoprim/sulfamethoxazole and tetracycline. These phenotypic data could be attributed to the presence of dfrA, sul, and tet genes (Table 3). The aadA, strA, and strB streptomycin resistance genes were also frequently detected among the 3 groups of 3GCs-R-Ec isolates. An association between those antibiotic resistance genes, the class I and class II integrase genes intI1 and intI2, and 3GC resistance was mainly observed in the isolates that contained blaCTX-M-1 and blaSHV-12 and only in single isolates that contained blaCMY-2 and blaTEM-52. Indeed, of 12 CMY-2– and 3 TEM52–positive isolates, 9 and 2, respectively, did not contain any additional resistance genes (Table 3). The absence of resistance to

(Büttner et al., 2011). The prevalence and its 95% confidence interval were calculated using the online confidence interval calculator (http://www.measuringusability.com). 3. Results and discussion 3.1. Prevalence of 3GC-R E. coli isolates

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Fig. 1. The phylogenetic tree constructed from the pulsed-field gel electrophoresis (PFGE) pattern of the 34 third-generation cephalosporin-resistant (3GCs-R) E. coli isolates found in broilers (October 18, 2010–April 30, 2011), swine, and cattle (January 1, 2011–April 30, 2011), including slaughterhouses (uppercase letters), geographic origin of the animals (cantons), resistance mechanisms, and sequence types (ST). Cantons: AG = Aarau; BE = Bern; FR = Fribourg; GE = Geneva; JU = Jura; LU = Lucerne; SG = St. Gallen; SH = Schaffhausen; SO = Solothurn; TG = Thurgau; VD = Vaud; ZH = Zurich; U = unknown origin.

other antibiotics other than β-lactam suggests that the CMY-2 and TEM-52 producers have likely been selected by the use of only βlactam antibiotics. On the other hand, the CTX-M-1 and SHV-12 producers could also have been selected by the use of other classes of antibiotics. The association of these genes might be related to the different epidemiologic evolution or sources of the CMY-2 producers versus the ESBL producers.

Interestingly, we noted that the majority of the isolates carrying CMY-2 fell into 1 PFGE cluster, which predominantly contained ST61. These isolates were found in broilers that were raised in different cantons and in various slaughterhouses. On the other hand, the CTXM–type ESBLs were carried by heterogeneous clones of E. coli, as shown by the numerous PFGE profiles and STs found.

3.5. Conclusions 3.4. Analysis of clonality The results of the PFGE and MLST analyses along with the epidemiologic data are presented in Fig. 1. Overall, many different PFGE profiles and STs were found. It should be noted that we did not find 3GC-R-Ec isolates of ST43, an ST that corresponds to ST131 when using the MLST databases of the University College Cork (http://mlst. ucc.ie/mlst/dbs/Ecoli) and that is widespread worldwide. In this regard and in order to determine the association between 3GCs-R and virulence factors, the detection of virulence genes should also be envisaged in survey programs studying the epidemiologic spread of 3GC-R-Ec in animals and humans.

In this work, we provide a representative nationwide prevalence and the molecular characteristics of 3GCs-R-Ec that were found among swine, cattle, and broilers at slaughterhouses. The most remarkable finding of this study was the high prevalence of CMY-2– producing E. coli isolates, their relative susceptibility to other antibiotic classes, and the association of the majority of the CMY-2 producers to a specific clonal lineage. Based on the associated resistance gene patterns and clonal analyses, we speculate that CMY-2–producing E. coli isolates might have a relatively recent and common source of origin, whereas those producing ESBLs are probably already widespread among animals, rendering their control

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very difficult. We also hypothesize that the presence of CMY-2 as a single antibiotic-resistance trait has probably been selected by the use of β-lactam antibiotics. It is therefore necessary to limit the use of antibiotics in food-producing animals and to implement hazard analysis critical control point procedures throughout the entire production to facilitate the early detection of 3GCs-R-Ec–positive animals in breeding farms and hatcheries and to avoid the spread of positive animals in different husbandries. Acknowledgments The authors thank the platform Genotyping of Pathogens and Public Health (Institut Pasteur) for coding MLST alleles and profiles. The authors also thank the participating slaughterhouses and their personnel for the sampling and acquisition of data, as well as Werner Lehmann, Isabelle Bertschy, and Andreas Thomann for assistance. References Batchelor M, Hopkins KL, Liebana E, Slickers P, Ehricht R, Mafura M, et al. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int J Antimicrob Agents 2008;31: 440–51. Blanc V, Mesa R, Saco M, Lavilla S, Prats G, Miro E, et al. ESBL- and plasmidic class C βlactamase-producing E. coli strains isolated from poultry, pig and rabbit farms. Vet Microbiol 2006;118:299–304. Bogaerts P, Hujer AM, Naas T, de Castro RR, Endimiani A, Nordmann P, et al. Multicenter evaluation of a new DNA microarray for rapid detection of clinically relevant bla genes from β-lactam-resistant Gram-negative bacteria. Antimicrob Agents Chemother 2011;55:4457–60. Bush K, Fisher JF. Epidemiological expansion, structural studies, and clinical challenges of new β-lactamases from Gram-negative bacteria. Annu Rev Microbiol 2011;65: 455–78. Büttner S, Flechtner O, Müntener C, Kuhn M, Overesch G. Bericht über den Vertrieb von Antibiotika in der Veterinärmedizin und das Antibiotikaresistenzmonitoring bei Nutztieren in der Schweiz (ARCH-VET 2009). Bern, Switzerland: Federal Veterinary Office and Swissmedic; 2010. Available at: http://www.swissmedic. ch/archvet-d.asp. Büttner S, Flechtner O, Müntener C, Overesch G. Bericht über den Vertrieb von Antibiotika in der Veterinärmedizin und das Antibiotikaresistenzmonitoring bei Nutztieren in der Schweiz (ARCH-VET 2010). Bern, Switzerland: Federal Veterinary Office and Swissmedic; 2011. Available at: http://www.swissmedic.ch/archvet-d.asp. Carattoli A. Animal reservoirs for extended spectrum β-lactamase producers. Clin Microbiol Infect 2008;14(Suppl 1):117–23. Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard - eighth edition, M07-A8. Wayne, PA: CLSI; 2009. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: 21st information supplement (M100-S21). Wayne, PA: CLSI; 2011. Coque TM, Baquero F, Canton R. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill 2008;13:1–11. Cortés P, Blanc V, Mora A, Dahbi G, Blanco JE, Blanco M, et al. Isolation and characterization of potentially pathogenic antimicrobial-resistant Escherichia coli strains from chicken and pig farms in Spain. Appl Environ Microbiol 2010;76: 2799–805. Dierikx C, Essen-Zandbergen A, Veldman K, Smith H, Mevius D. Increased detection of extended spectrum β-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry. Vet Microbiol 2010;145:273–8. Doi Y, Paterson DL. Detection of plasmid-mediated class C β-lactamases. Int J Infect Dis 2007;11:191–7. Dolejska M, Duskova E, Rybarikova J, Janoszowska D, Roubalova E, Dibdakova K, et al. Plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli isolates from an equine clinic and a horseback riding centre. J Antimicrob Chemother 2011;66: 757–64. Endimiani A, Bertschy I, Perreten V. Escherichia coli producing CMY-2 β-lactamase in bovine mastitis milk. J Food Protect 2011;75:137–8. Endimiani A, Doi Y, Bethel CR, Taracila M, Adams-Haduch JM, O'Keefe A, et al. Enhancing resistance to cephalosporins in class C β-lactamases: impact of Gly214Glu in CMY-2. Biochemistry 2010;49:1014–23. Endimiani A, Hujer AM, Hujer KM, Gatta JA, Schriver AC, Jacobs MR, et al. Evaluation of a commercial microarray system for detection of SHV-, TEM-, CTX-M-, and KPC-type β-lactamase genes in Gram-negative isolates. J Clin Microbiol 2010;48:2618–22.

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