AAC Accepts, published online ahead of print on 16 September 2013 Antimicrob. Agents Chemother. doi:10.1128/AAC.01460-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

1    1 

Shor t-for m paper

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National pr evalence of r esistance to thir d-gener ation cephalospor in in E. coli isolated

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fr om Fr ench layer flocks

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Running title: resistance to third-generation cephalosporin in layers

5 

6 

Claire Chauvin 1,2, Laetitia Le Devendec1,2, Eric Jouy1,2, Maena Le Cornec1,2, Sylvie

7 

Francart3, Corinne Marois-Créhan1,2 and Isabelle Kempf 1,2

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1

ANSES, Ploufragan Laboratory, 22440 Ploufragan, France

9 

2

Université Européenne de Bretagne, Rennes, France

10 

3

Direction Générale de l’Alimentation, Ministère de l’Agriculture et de l’Agro-alimentaire,

11 

251, rue de Vaugirard, 75732 Paris Cedex 15, France

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*Corresponding author: Isabelle Kempf, ANSES, Ploufragan Laboratory, Mycoplasmology-

13 

Bacteriology Unit, 22440 Ploufragan, France

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Phone: 33 2 96 01 62 81

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Fax: 33 2 96 01 62 73

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Email: [email protected]

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2    18 

19 

Abstr act

20 

Resistance to third-generation cephalosporin (3GC) of E. coli in fecal samples representative

21 

of the French egg production was studied. The susceptibility to cefotaxime of E. coli isolates

22 

obtained by culture on non-selective media was determined. Twenty-two non-susceptible

23 

isolates were obtained (7.51% [4.49-10.54]), in majority from young birds. Most isolates

24 

carried a bla CTX-M-1 group gene and a few ones carried a bla CMY-2-like gene. Control of 3GC

25 

resistance in laying hens is needed.

3    26 

27 

Several studies have reported the high prevalence of resistance to third-generation

28 

cephalosporin (3GC) in broilers in different countries (1-4) but little is known about

29 

prevalence in the egg production sector. Thus fecal samples collected from French pullets and

30 

laying hens, in the framework of the official national plan for the control of Salmonella

31 

infection, were used to evaluate the prevalence of E. coli not susceptible to 3GC.

32  33 

From February to May 2011, 300 pools of fresh fecal samples were collected by veterinary

34 

services from 300 different production flocks (283 different farms) in 71 French

35 

départements, according to official instructions.

36 

For each sample, one E. coli isolate, obtained on MacConkey media, was randomly collected

37 

and identified (5). A standardized inoculum of each isolate was deposited on Mueller Hinton

38 

agar containing cefotaxime (1 mg/L) (6). The isolates with a MIC of cefotaxime higher than 1

39 

mg/L were further analyzed. Their susceptibility to other antimicrobials was tested by the disk

40 

diffusion method (7) and determination of MIC using Sensititre plates (Biocentric, Bandol,

41 

France). Phylogenetic groups were determined (8) and pulsed-field gel electrophoresis

42 

(PFGE) was performed after restriction with XbaI (9). The presence of conjugative plasmids

43 

was checked by conjugation. Plasmids were prepared (10) and used to transform E. coli

44 

DH5alpha. 3GC resistance genes of the isolates were detected by use of microarrays (Check-

45 

MDR-CT101, Biocentric) (11) and sequencing (12, 13). The replicon types were identified

46 

(14).

47 

Among 293 viable E. coli, 22 (7.51% [4.49-10.54]) isolates could grow on cefotaxime-

48 

supplemented agar. Non-susceptible isolates were more frequently obtained from young birds

49 

and isolation probability decreased weekly (P=0.0002; OR=0.94 [0.91-0.97]) (Figure 1).

4    50 

Similarly, non-susceptible isolates were more frequent in pullets than in layers ((P64mg/L. The PFGE profiles of the 14 typeable strains all appeared

58 

different from each other. Conjugation and transformation experiments revealed that

59 

resistance to cephalosporins, and most often to tetracycline or trimethoprim-sulfamethoxazole

60 

could be transferred to recipient cells. The replicons identified in the transformants were

61 

IncI1. The 3GC resistance genes belonged to the bla CTX-M-1 group for all strains. Profiles

62 

obtained after EcoRI digestion of plasmid DNA revealed that similar profiles could be

63 

obtained for plasmids obtained from strains 2 and 110, from strains 40, 96 and 112 and from

64 

strains 273 and 294 (data not shown).

65 

For 5/22 isolates, all obtained from pullets, no synergy between amoxicillin- clavulanic acid

66 

and 3GC could be observed; these five isolates were resistant to cefoxitin. They also exhibited

67 

additional resistance to tetracycline, and four of them exhibited resistance to aminoglycosides.

68 

All were highly resistant to ceftriaxone (MIC>8mg/L), except strain 260 for which the

69 

ceftriaxone MIC was 2mg/L. The four typeable strains showed profiles that were different

70 

from each other. Conjugation and transformation experiments indicated that resistance to

71 

cefoxitin could be transferred from the four strains that were highly resistant to ceftriaxone,

72 

sometimes with other resistances (Table 1). The replicons associated with transfer of cefoxitin

73 

resistance were IncI1, B/O, A/C or F. Four strains and their transformants contained a bla CMY-

5    74 

2-like

gene. The electrophoresis profiles of the EcoRI-digested plasmids obtained from the

75 

transformants of isolates 121 and 134 were similar to each other (data not shown).

76  77 

Very limited data concerning 3GC resistance in the egg production sector are available (15),

78 

except for one involving a few organic production farms in Denmark (16). Wasyl et al.

79 

revealed that 42.3% of 163 samples from layers in Poland and other European countries,

80 

contained 3GC- resistant E. coli, when inoculated on cefotaxime-supplemented media, a

81 

sensitive procedure that enables the detection of rare resistant isolates among many non-

82 

resistant E. coli (16). When isolates from non-supplemented media were tested, the proportion

83 

of resistant isolates was only 0.6%. The higher percentage obtained in our study probably

84 

reflects the fact that we sampled pullets and layers of all ages and not only birds arriving at

85 

the slaughterhouse, which, as discussed below, less frequently carry 3GC-resistant E. coli.

86 

Our results showed that resistant E. coli were more often isolated from pullets than from

87 

laying hens and that the proportion of resistant isolates varied in samples obtained from birds

88 

from different hatcheries. It is tempting to speculate that this could be related to antimicrobial

89 

treatments of young birds or off-label use of ceftiofur in hatcheries (e.g. in ovo or

90 

subcutaneous injection) (17, 18). Moreover vertical transmission and circulation of 3GC-

91 

resistant isolates in farms and hatcheries are probable in meat poultry (19) and egg (20)

92 

productions. It is also plausible that administration of frequently-prescribed molecules

93 

(tetracycline or trimethoprim-sulphonamides) results in co-selection of 3GC resistance.

94 

As in broilers in France (1, 21), the most prevalent resistance gene was bla CTX-M- 1/61 borne on

95 

an IncI1 plasmid. The dissemination of bla CTX-M- 1 on IncI1 plasmids has also been observed

96 

in E. coli and in Salmonella spp. in a number of countries, in poultry and in other animal

97 

species (4), but characterization by pMLST revealed that different plasmids may be present

6    98 

(22-25). The other gene detected in pullets was bla CMY-2/22/61, which is also frequent in poultry

99 

in other countries (26-30).

100 

It is feared that ESBL genes might spread from poultry to humans either through professional

101 

exposure (23) or via food. In this context, it is interesting to underline that bla CTX-M-1 was,

102 

along with bla CTX-M-15, one of the two most frequent ESBL genes harbored by E. coli in fecal

103 

samples obtained from healthy subjects in a Parisian check-up centre (31). Moreover the

104 

transfer in the hen gut, of plasmids encoding for resistance to 3GC from E. coli to Salmonella

105 

spp (32) could lead to problematic human infections with 3GC-resistant Salmonella-infected

106 

eggs. Therefore the monitoring and control of the presence of 3GC-resistant E. coli and

107 

Salmonella in egg production is essential.

108  109 

Acknowledgments

110 

This study was supported by the French Ministry of Agriculture and the Côtes d’Armor

111 

General Council. We are very grateful to M. Llagostera, UAB, Barcelona, Spain, for

112 

providing us with E. coli strain HB101 UA6190, A. Carattoli for providing reference strains

113 

and protocols for replicon typing and departmental veterinary laboratories for providing the E.

114 

coli isolates.

115 

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Garcia Fernandez A., Chiaretto G., Bertini A., Villa L., Fortini D., Ricci A., Carattoli A. 2008.  Multilocus sequence typing of IncI1 plasmids carrying extended spectrum beta lactamases in  Escherichia coli and Salmonella of human and animal origin. J. Antimicrob. Chemother.  61:1229 1233.  Leverstein van Hall M. A., Dierikx C. M., Cohen Stuart J., Voets G. M., van den Munckhof M.  P., van Essen Zandbergen A., Platteel T., Fluit A. C., van de Sande Bruinsma N., Scharinga J.,  Bonten M. J., Mevius D. J. 2011. Dutch patients, retail chicken meat and poultry share the  same ESBL genes, plasmids and strains. Clin. Microbiol. Infect. 17:873 880.  Endimiani A., Rossano A., Kunz D., Overesch G., Perreten V. 2012. First countrywide survey  of third generation cephalosporin resistant Escherichia coli from broilers, swine, and cattle in  Switzerland. Diag. Microbiol. Infect. Dis. 73:31 38.  Mnif B., Ktari S., Rhimi F. M., Hammami A. 2012. Extensive dissemination of CTX M 1  and  CMY 2 producing Escherichia coli in poultry farms in Tunisia. Letters in Appl. Microbiol.  55:407 413.  Smet A., Martel A., Persoons D., Dewulf J., Heyndrickx M., Catry B., Herman L.,  Haesebrouck F., Butaye P. 2008. Diversity of extended spectrum  lactamases and class C  lactamases among cloacal Escherichia coli isolates in Belgian broiler farms. Antimicrob.  Agents Chemother. 52:1238 1243.  Park Y. S., Adams Haduch J. M., Rivera J. I., Curry S. R., Harrison L. H., Doi Y. 2012.  Escherichia coli producing CMY 2  lactamase in retail chicken, Pittsburgh, Pennsylvania,  USA. Emerg. Infect. Dis. 18:515 516.  Xia L. N., Tao X. Q., Shen J. Z., Dai L., Wang Y., Chen X., Wu C. M. 2011. A survey of  lactamase and 16S rRNA methylase genes among fluoroquinolone resistant Escherichia coli  isolates and their horizontal transmission in Shandong, China. Foodborne Pathogens Dis.  8:1241 1248.  Nicolas Chanoine M. H., Gruson C., Bialek Davenet S., Bertrand X., Thomas Jean F., Bert F.,  Moyat M., Meiller E., Marcon E., Danchin N., Noussair L., Moreau R., Leflon Guibout V.  2013. 10 Fold increase (2006 11) in the rate of healthy subjects with extended spectrum  beta lactamase producing Escherichia coli faecal carriage in a Parisian check up centre. J.  Antimicrob. Chemother. 68:562 568.  Cloeckaert A., Praud K., Lefevre M., Doublet B., Pardos M., Granier S. A., Brisabois A., Weill  F. X. 2010. IncI1 plasmid carrying extended spectrum lactamase gene blaCTX M 1 in  Salmonella enterica isolates from poultry and humans in France, 2003 to 2008. Antimicrob.  Agents Chemother. 54:4484 4486. 

9    215 

TABLE 1 Characteristics of isolates Non-beta-

Départemen Age Strain t

(weeks) D10

resistance

Phylogenetic group

2

2 40

Non-beta-lactam

lactam

pAmpC

resistance

enzymes

transferred

CTX-M-

TET

TET KAN

A

1a

B1

CTX-M-1

D12

17

TET SXT

D11

24

TET NAL SXT

91

ESBL or

Replicon type c

I1 F SXT

I1 FIC F

SXT

STR

A

CTX-M-1

I1 F B/O

96

D3

ND

TET

B1

CTX-M-1

TET

I1 FIB F

110

D10

4

TET

A

CTX-M-1

TET

I1

112

D10

55

TET

A

CTX-M-1

TET

I1 FIB F

114

D10

59

TET

D

CTX-M-1

TET

I1 F

119

D10

4

TET

A

CTX-M-1

TET

I1 FIB

10   

125

D9

7

TET

A

CTX-M-1

TET

I1

180

D13

30

TET

B1

CTX-M-1

TET

I1 FIB F

195

D7

32

-

D

CTX-M-1

-

I1 FIB F

232

D2

21

TET CIP NAL

D

CTX-M-1

TET

I1 FIB F

241

D2

65

TET

B2

CTX-M-1

TET

I1 FIB F

270

D1

15

TET

D

CTX-M-1

TETd

I1

272

D1

5

TET KAN

D

CTX-M

TET

I1 FIB F

273

D1

9

TET

B1

CTX-M-1

TET

I1 FIB F B/O

294

D4

13

TET

D

CTX-M-1

TET

I1 FIB F

121

D10

8

TET KAN STR

D

CMY-2b

-

K B/O

134

D6

4

TET STR

A

CMY-2

TET

I1 A/C

174

D8

6

TET STR

A

CMY-2

TET

A/C K B/O

192

D8

15

TET

A

CMY-2

-

I1 FIB F

260e

D1

11

TET KAN STR

B1

ND

-

I1 F

11   

sequence compatible with bla CTX-M- 1/61; bsequence compatible with bla CMY-2/22/61; cco-transferred by conjugation or by transformation; d no

216 

a

217 

transformant could be obtained for isolate 270; etransfer of the 3GC resistance could not be obtained by conjugation or transformationfor isolate

218 

260

219  220 

ND: not determined

12    221 

Figur e 1 Distribution of samples according to age of flock (in weeks) and results for susceptibility to third-generation cephalosporins (France

222 

2011, 293 samples)

223 

S: susceptible; Non-S: non-susceptible

224 

(One non-susceptible strain was obtained from a sample with no indication of age)

225  226  227  228