Journal of Antimicrobial Chemotherapy Advance Access published July 28, 2014
J Antimicrob Chemother doi:10.1093/jac/dku270
blaCTX-M-15-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany
1
Federal Institute for Risk Assessment, National Reference Laboratory for Antimicrobial Resistance, Department Biological Safety, Max-Dohrn Straße 8-10, D-10589 Berlin, Germany; 2Hospital Universitario Ramo´n y Cajal, Servicio de Microbiologı´a, Madrid, Spain; 3 Barcelona Centre for International Health Research (CRESIB), Hospital Clı´nic—Universidad de Barcelona, Barcelona, Spain; 4Robert Koch Institute, FG13 Nosocomial Pathogens and Antimicrobial Resistance, Wernigerode, Germany *Corresponding author. Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany. Tel: +49-30-18412-2082; Fax: +49-30-18412-2953; E-mail: [email protected]
Received 4 December 2013; returned 18 January 2014; revised 10 June 2014; accepted 21 June 2014 Objectives: The characterization of CTX-M-15 b-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. Methods: E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to thirdgeneration cephalosporins, originating from routine and monitoring submissions (2003 – 12) to the Federal Institute for Risk Assessment and different national targeted studies (2011–12), were examined for the presence of blaCTX-M-15 genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-15 were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. Results: Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-15 producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-15 genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. Conclusions: The prevalence of CTX-M-15-producing isolates from non-human sources in Germany is still low. The blaCTX-M-15 gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-15 gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-15-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance. Keywords: antimicrobial resistance, CTX-M, ESBLs, plasmids, chromosomal encoded
Introduction Worldwide the number of bacteria resistant to third- and fourthgeneration cephalosporins has increased (http://www.ecdc.europa. eu/en/activities/surveillance/EARS-Net).1 In most cases resistance is caused by the production of extended-spectrum b-lactamases (ESBLs) or AmpC-type b-lactamases. In Enterobacteriaceae, members of the CTX-M family are the predominant ESBLs.2 The b-lactamase blaCTX-M genes are in most cases located on transmissible genetic elements such as plasmids and transposable elements including insertion sequences (ISs) such as ISEcp1.3 The spread of
the blaCTX-M-15 gene, which encodes the most widely distributed CTX-M enzyme in Enterobacteriaceae of human origin,1,2,4 – 6 is mainly related to the rapid dissemination of the highly virulent extraintestinal pathogenic Escherichia coli clone O25:H4-B2-ST131 (implicated in human urinary tract and nosocomial infections).7 – 9 However, none of the ESBL-ST131-positive isolates from livestock or foods described so far has been a CTX-M-15 producer.1,4,9,10 In livestock CTX-M-1 is the most frequent ESBL in E. coli and Salmonella, and only sporadic occurrences of CTX-M-15producing E. coli in swine, cattle and poultry isolates have been described.1,4,9 – 11
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Jennie Fischer1, Irene Rodrı´guez1,2, Beatrice Baumann1, Elisabeth Guiral1,3, Lothar Beutin1, Andreas Schroeter1, Annemarie Kaesbohrer1, Yvonne Pfeifer4, Reiner Helmuth1 and Beatriz Guerra1*
Fischer et al.
Materials and methods Bacterial isolates, b-lactam susceptibility testing and detection of ESBL/AmpC-encoding genes In total, 677 non-human E. coli (526) and Salmonella (151) isolates originating mainly from food and healthy food-producing animals (including 4 E. coli and 14 Salmonella isolates from companion and wild animals, feed, manure and a toy) were analysed. Of these isolates, 418 (270 E. coli and 148 Salmonella collected from January 2003 to July 2012 and submitted to the National Reference Laboratories NRL-AR, NRL-Salm and NRL-E. coli of the BfR) originated from routine diagnostic and national monitoring and surveillance programmes (i.e. commensal E. coli, verotoxin-producing E. coli and Salmonella; swine, cattle, turkey, poultry and their respective meats) and showed MIC values of cefotaxime or ceftazidime above the EUCAST epidemiological cut-offs (0.25 and 0.5 mg/L, respectively; http://www.eucast.org). The other 259 isolates (256 E. coli and three Salmonella) were collected using MacConkey agar supplemented with 1 mg/L cefotaxime as a selective medium in different targeted studies [66 swine, poultry and cattle farms; January 2011 and July 2012; Table S1 (available as Supplementary data at JAC Online)] within the national RESET Project (www.reset-verbund.de). All isolates were tested for their susceptibility to 17 b-lactams by the disc diffusion method (CLSI M2-A10) and for the presence of ESBL/ AmpC-encoding genes by PCR amplification/sequencing as previously described.6,11,14 For comparison, three human control strains were investigated.
Typing of CTX-M-15-producing isolates and characterization of antimicrobial resistance and virulence determinants Isolates positive for blaCTX-M-15 (23) were analysed by serotyping, XbaI PFGE (www.pulsenetinternational.org), multilocus sequence typing (MLST; http://mlst.warwick.ac.uk/) and phylogenetic grouping (E. coli) as previously described.6,11 The resistance phenotypes of the isolates were determined by testing their antimicrobial susceptibility to 14 antimicrobials by broth microdilution (CLSI M7-A8) (http://www.bfr.bund.de/cm/350/german-antimicrobialresistance-situation-in-the-food-chain-2009.pdf). The presence of additional resistance genes and integrons, as well as virulence genes, was screened using DNA microarrays (E. coli Genotyping Kit, Alere Technologies GmbH, Jena, Germany) following the manufacturer’s recommendations (http://alere-technologies.com/fileadmin/Media/Downloads/op/50202/ 05_16_04_0006_V05_Manual_E.coli.pdf) and/or PCR amplification/sequencing.6,11 The expression of the b-lactamase genes was tested by isoelectric focusing.6 Linkage of ISEcp1 with the blaCTX-M-15 genes was analysed by PCR amplification/sequencing.11 Plasmids were isolated by alkali lysis and transformed into E. coli DH10B Competent Cells.6,11 The plasmid content of the CTX-M-15-producing isolates and transformants (selected on MacConkey agar plates supplemented with 1 mg/L cefotaxime) was visualized by S1 PFGE.6,11 The location of the blaCTX-M-15 gene was tested by Southern blot
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hybridization of the S1, XbaI PFGE and Kado plasmid gels with a blaCTX-M-15 probe.6,11 Incompatibility (Inc) groups of plasmids were determined by PCR replicon typing (PBRT kit, Diatheva, Fano, Italy). IncI1 and IncF plasmids were analysed by plasmid MLST (pMLST; www.pubmlst.org/ plasmid/, accessed 18 July 2013). Conjugation experiments of selected blaCTX-M-15-carrying ST410 isolates (10 isolates) were performed by filter mating at 378C and room temperature.6,11
Results and discussion Among the 677 third-generation cephalosporin-resistant isolates analysed (526 E. coli and 151 Salmonella isolated mainly from food and healthy food-producing animals until July 2012), only 23 CTX-M-15-producing isolates (21 E. coli from livestock, food and a toy and 2 Salmonella from animals; 4.0% and 1.3%, respectively) were found (Table 1). The first CTX-M-15-producing E. coli isolated from livestock (Table 1) was collected in 2010, suggesting a recent emergence of this gene in the food-producing animal E. coli population in Germany. As described by other authors,1,4,9,10,15,16 also in bacteria from livestock and foods in Germany, the prevalence of blaCTX-M-15 is still low, in contrast to the high prevalence of E. coli with blaCTX-M-15 in humans in Germany.5,17 In our series, however, this prevalence varied among the studies in which isolates were collected, as shown in Table S1. To investigate whether the presence of CTX-M-15 producers was related to clonal spread or to the location of blaCTX-M-15 on mobile genetic elements, the molecular properties of the 21 E. coli and the two Salmonella isolates were analysed (Table 1 and Figure 1). MLST revealed that none of the E. coli isolates belonged to the previously mentioned E. coli O25:H4-B2-ST131, excluding the possible spread of this clone as a source for CTX-M-15-producing isolates in livestock and foods in Germany. On the other hand, three of the isolates were epidemiologically related to the German E. coli O104:H4-B1-ST678-blaCTX-M-15 outbreak strain and were recovered from an active surveillance of foods and patient’s households (Table 1 and Figure 1). After the outbreak in 2011, E. coli O104: H4-B1-ST678 isolates were rarely found in humans, and they had never previously been detected in food or animals.18,19 These isolates were highly pathogenic, characterized by the presence of several genes associated with enteroaggregative and enterohaemorrhagic E. coli (EHEC) strains as well as multidrug resistance.12,13,19 The isolates carried the blaCTX-M-15 gene on an 80 kb IncI1-pST31 plasmid (Table 1). IncI1 plasmids, in particular IncI1-pST31 and pST37 (http://pubmlst.org/; access date 28 June 2013; both plasmid types present in other isolates of the series), play an important role in the spread of blaCTX-M-15 in both humans and animals.3,10,15 A second detected clone comprised four E. coli isolates originating from turkey meat and swine (collected between January and May 2012) and ascribed to ST167/ST617, clonal complex CC10 (Table 1 and Figure 1). ESBL-producing E. coli isolates with these STs are commonly reported from humans but seldom from animals.4,9,10 The four isolates carried the blaCTX-M-15 genes on large multireplicon IncFIA/FIB/FII plasmids. These IncF plasmids carried additional genes [blaOXA-1, aac(6′ )-1b-cr or class 1 integrons; Table 1] characteristic of O25:H4-B2-ST131 IncF plasmids.3,7 Other E. coli blaCTX-M-15 isolates detected in the series also belonged to ST types (ST117, ST354, ST162 and ST196) commonly found among human E. coli isolates (http://mlst.ucc.ie/mlst/dbs/).
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In 2011 an enteroaggregative Shiga-toxin-producing E. coli (EAEC-STEC) O104:H4-B1-ST678 strain producing CTX-M-15 caused one of the severest foodborne outbreaks in Germany and Europe, affecting more than 3842 people.12,13 To look for a possible reservoir of this strain in animals or foods and to ascertain a potential relationship between the spread of CTX-M-15 producers in humans, livestock and food, we characterized all non-human CTX-M-15-producing E. coli and Salmonella isolates from the strain collections of the Federal Institute for Risk Assessment (BfR).
ALA3/ALA4 amplicon
Resistance Federal state Isolate no.
Species
05E00174f
E. coli
Source
phenotype/
(isolation date) resistance genotype
human
B (12/04/05)
(infection)
[AMP CTX CAZ]* CIP
Serotypea O25:H4
Phylogroupb B2
PFGE
MLST (clonal
pattern
complex)
E-X1
Inc Size
group
(ISEcp1 pMLST
ST131 (none) 110 kb FIA, FII F2:A1
sequence)c
Class 1 integrond
—
1700 bp/
NAL KAN STR SMX
Virulence gene(s)e iha, nfaE, prfB, sat
dfrA17-aadA5
TET TMP/ [blaCTX-M-15 blaOXA-1], aac(6 ′ )-Ib-cr, [aadA5, strA/B], [sul1, sul2], tet(A), dfrA17 12E00226
E. coli
meat, turkey
NRW (14/ 03/12)
[AMP CTX CAZ]* CIP
O1:Hr
D
E-X7
NAL SMX/
noneg
—
ST117 (none) 80 kb
I1
ST354
383 bph
—
lpfA
383 bph
—
lpfA, prfB, pic, vat, cba,
(CC354)
blaCTX-M-15, sul2 11E00851
E. coli
cattle
NRW (06/
(commensal)
06/11)
[AMP CTX CAZ]* CIP
ONT:HNT
D
E-X3
ST31
NAL STR SMX TET
cma, mcmA
TMP/[blaCTX-M-15, blaTEM-1], strA/B, sul2, tet(A), dfrA14 12E00587
E. coli
meat, calf
NRW (03/ 05/12)
[AMP CTX CAZ]* CHL
O55:HNT (fliCH10)
B1
E-X8
CIP NAL GEN KAN
ST162
85 kb
I1
ST37
383 bpi
—
lpfA, mchB, mchC, mchF
(CC469)
STR SMX TET TMP/ [blaCTX-M-15, blaTEM-1], catA1, aphA1, [aadA5, strA/B], sul2, tet(B), dfr17 11E00604
E. coli
cattle
NI (20/04/11)
(commensal)
[AMP CTX CAZ]* TET/
O8:H7
B1
E-X2
ST196 (none) 80 kb
I1
ST31
383 bph
—
f17-G, lpfA, cdtB, cnf1
O104:H4
B1
E-X6
ST678 (none) 80 kb
I1
ST31
383 bph
700 bp/dfrA7
stx2a, aggA, aap, aatA,
blaCTX-M-15 in non-human German Escherichia coli and Salmonella
Table 1. Phenotypic and molecular features of CTX-M-15-producing non-human enterobacterial isolates from Germany, 2005 –12
[blaCTX-M-15, blaTEM-1], tet(A)
O104 outbreak
E. coli
n ¼5 (NRZ-12-2027f, f
11E03787 ,
human (2,
ST (2), HE, NI
[AMP CTX CAZ]* STR
infection),
(2) (06-15/
SMX TET TMP/
aggR, pic, set1iha,
salmon,
06/11)
[blaCTX-M-15,
fyuA, irp2, terB, terD k
blaTEM-1], strA/B,
cucumber, toy
11E03789j,
[sul1, sul2],
11E03786j and
tet(A), dfrA7
11E03794j ) R363
E. coli
swine
BY (25/04/12)
(commensal)
[AMP CTX CAZ]* CIP
ONT:H10
A
E-X10
ST617 (CC10) 170 kb FII,
F31:A4:B1
NAL GEN SMX TET
FIA,
TMP/[blaCTX-M-15,
FIB
383 bph
1700 bp/
(astA)
dfrA17-aadA5
blaOXA-1], aac(6 ′ )-Ib-cr, aadA5, [sul1, sul2], tet(B), dfrA17
Continued
JAC
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ALA3/ALA4 amplicon
Resistance Federal state Isolate no. 12E00937
Species E. coli
Source
phenotype/
(isolation date) resistance genotype
meat, turkey
NRW (23/ 05/12)
[AMP CTX CAZ]* CIP
Serotypea ONT:HNM
Phylogroupb A
PFGE
MLST (clonal
pattern
complex)
E-X9
Inc Size
group
(ISEcp1 pMLST F31:A4:B37l
ST167 (CC10) 140 kb FII,
NAL STR SMX TET/
FIA,
[blaCTX-M-15,
FIB
sequence)c
Class 1 integrond
383 bph
—
Virulence gene(s)e Iha, sfaS, (espA_C_ rodentium), senB
blaOXA-1], aac(6 ′ )-Ib-cr, strA/B, sul2, tet(A) 12E00276
E. coli
meat, turkey
NRW (04/ 04/12)
[AMP CTX CAZ]* CIP
ONT:HNM
A
E-X5b
ST167 (CC10) 170 kb FII,
F31:A4:B1
NAL STR SMX TET
FIA,
TMP/[blaCTX-M-15,
FIB
383 bph
1700 bp/ dfrA17-aadA5
blaOXA-1], aac(6 ′ )-Ib-cr, [aadA5, strA/B], tet(A), dfrA17 R261
E. coli
swine
BB (09/01/12)
(commensal)
[AMP CTX CAZ]* CIP
ONT:H9
A
E-X5a
ST167 (CC10) 145 kb FII,
F31:A4:B1
NAL KAN STR SMX
FIA,
TET/[blaCTX-M-15,
FIB
383 bph
—
—
lpfA, cma
blaOXA-1], aac(6 ′ )-Ib-cr, strA/B, sul2, tet(A) R208
E. coli
swine
NRW (29/
(commensal)
06/11)
[AMP CTX CAZ]* CIP
O8:H9
A
E-X4d
ST410 (CC23) noneg
—
382 bph
—
Or:H9
A
E-X4a
ST410 (CC23) noneg
—
382 bph
1600 bp/dfrA1-
NAL TET/ blaCTX-M-15, [tet(A), tet(B)]
10E00080
E. coli
cattle
NI (15/02/10)
(commensal)
[AMP CTX CAZ]* CHL CIP NAL STR SMX
aadA1;
TET TMP/
1900 bp/
[blaCTX-M-15,
drfA12-aadA2
cma
blaTEM-1], catA1, [aadA1, aadA2, strA/B], [sul1, sul3], tet(B), [dfrA1, drfA12] R107
E. coli
cattle
NRW (29/
(commensal)
06/11)
[AMP CTX CAZ]* CIP
O8:HNT (fliCH9)
A
E-X4b
ST410 (CC23) noneg
382 bph
—
NAL STR SMX TET
1900 bp/
lpfA, cma
dfrA12-aadA2
TMP/blaCTX-M-15, aadA2, sul1, tet(A), dfrA12 E. coli
swine (commensal)
ST410 (CC23) 110 kbg FII,
NRW (24/
[AMP CTX CAZ]* CHL
05/11)
CIP NAL STR SMX
FIA,
TMP/[blaCTX-M-15,
FIB
O20:H9
A
E-X4e
F31:A4:B1
382 bph
1700 bp/ dfrA17-aadA5
blaOXA-1], aac(6 ′ )-Ib-cr, [aadA5, strA/B], [sul1, sul2], dfrA17
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R37
(lpfA, sfaS), senB
Fischer et al.
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Table 1. Continued
E. coli
swine (3), poultry NRW (n ¼4, 14/ [AMP CTX CAZ]* CHL
ONT:HNM (fliC
R61a, R283,
(2), cattle (1)
06/12-04/
CIP NAL FFN GEN
H21) (5),
R341 and R345)
(commensals)
07/11), BY
KAN STR SMX TET
ONT:H21
(23/04/12),
TMP/blaCTX-M-15,
TH (17/
catA1, floR,
08/11)
[aadA5, strA/B]
A
E-X4c
ST410 (CC23) noneg
1172 bpm
—
1700 bp/ dfrA17-aadA5
f17-G, (f17-A)+ (lpfA, espB_O26, espA_C_rodentium; espP)
aadA5, sul1, tet(A), dfrA17 05-01901
Salmonella horse (infection)
HE (27/04/05)
[AMP CTX CAZ]* STR SMX TET TMP/
Salmonella
S-X1
not done
90 kb
I1
ST31
383 bph
800 bp/aadB; 1200 bp/
Typhimurium
[blaCTX-M-15,
blaPSE-1
blaTEM-1, blaPSE-1], aadB, sul1, tet(G) 09-1454
Salmonella swine
HE (17/04/09)
(commensal)
[AMP CTX CAZ]* CHL CIP GEN STR SMX
Salmonella
S-X2
not done
320 kb HI2
not done
383 bph
1000 bp/aadA1
Bredeney
TET TMP/ [blaCTX-M-15, blaTEM-1, blaOXA-1], aac(6 ′ )-Ib-cr, aadA1, strA, sul2, tet(A), dfrA1-like
Abbreviations. Antimicrobials: AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; FFN, florfenicol; KAN, kanamycin; NAL, nalidixic acid; SMX, sulfamethoxazole; STR, streptomycin; TET, tetracycline; TMP, trimethoprim. Regions: B, Berlin; BY, Bavaria; HE, Hesse; NI, Lower Saxony; NRW, North Rhine-Westphalia; ST, Saxony-Anhalt. Others: r, rough; NM, non-motile; NT, not typeable. MICs were determined using CLSI methodology (M07-A8). Evaluation of the results was performed according to the European Community Decision 2007/407/EC (http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2007:153:0026:0029:EN:PDF) using the EUCAST epidemiological cut-off values (www.eucast.org). All isolates were susceptible to colistin. [AMP CTX CAZ]*: MIC phenotype. These isolates showed microbiological resistance to the penicillins and first- to third-generation cephalosporins and monobactams tested, and susceptibility to cephamycins and carbapenems, as obtained by the disc diffusion method following the EUCAST guidelines and cut-off values (www.eucast.org) as previously described.11 a Serotyping of E. coli, performed as described in Beutin et al. 12 Serotyping of the Salmonella isolates performed following the White– Kauffmann –Le Minor scheme (www.pasteur.fr). b Performed as described by Rodrı´guez et al.6 c PCR amplicons obtained using the ISEcp1-related primer ALA3 and a modified ALA4.6 d Approximate size of the amplicon obtained using the 5′ CS-3′ CS primers/gene contained in the variable region.11 e Virulence genes were analysed with the E. coli Genotyping Kit (Alere Microarray). Genes tested and row results obtained are shown in Table S2. Genes in brackets were recognized only with an ambiguous signal in the array. f Reference strain (control). Strain no. 05E00174, from a urinary tract infection received by the NRL-E. coli, represented the E. coli clone O25:H4-B2-ST131-blaCTX-M-15. Strains EHEC O104 NRZ-12-2027, kindly provided by Dr Tietze (Robert Koch Institute), and 11E03787 (received by the NRL-E. coli) represented the E. coli O104:H4-B1-ST678-blaCTX-M-15 and were isolated from patients during the German outbreak in 2011. g Chromosomally located blaCTX-M-15 gene. h Identical to HF549093.1 sequence. i Sequence identical to the one with accession number GQ385309.1. j Isolates related to the German EHEC O104:H4-ST678 outbreak: salmon recovered from a catering service prepared by an intoxicated patient (secondary outbreak), cucumber recovered from the garbage of an intoxicated family, toy recovered from a patient household. k Characterized by Miko et al.19 l Differs from IncFIB 1 in only one nucleotide. m The tnpA gene is disrupted by the insertion of IS1 (HG798331).
blaCTX-M-15 in non-human German Escherichia coli and Salmonella
n ¼ 6 (R54, R56,
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Fischer et al.
(a) kb
CC10
ST678 Br 1
2
3
4
5
6 Br
7
8
ST410
9 10 11 12 13 14 15 Br
452.7
244.4 138.9
33.3
ST410
(b) kb
Br 1
2
3
4
5
6
ST678 7
8
9 10 Br 11 12 13 14 15 Br
1135
452.7
244.4
*
138.9
33.3
Br S. Braenderup H9812 1 - R208 2 - 10E0080 3 - R107 4 - R37 5 - R283 6 - R54 7 - R56 8 - R61 9 - R345 10 - R341 11 - O104 12 - 11E3786 13 - 11E3787 14 - 11E3789 15 - 11E3794
*
Figure 1. XbaI PFGE patterns from blaCTX-M-15-positive E. coli. (a) All different XbaI PFGE patterns. CC10 includes isolates of ST167 (lanes 8– 10) and 617 (lane 7). (b) XbaI PFGE patterns from E. coli isolates belonging to the A-ST410 clone (*280 kb band, which hybridized with a blaCTX-M-15 probe) or B1-ST678 clone.
One of the most interesting findings of the present work was the detection of the widespread E. coli ST410 phylogroup A clonal line. E. coli ST410 are frequently isolated from humans and animal sources and have also been recovered from human clinical samples.4,10,16,20 The isolates from the present study ascribed to this line (10) were collected from swine, cattle and poultry farms in four German federal states from February 2010 until July 2012 [Table 1, Figure 1 and Figure S1 (available as Supplementary data
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at JAC Online)]. These isolates were resistant to 13 of the 14 antimicrobials tested, retaining susceptibility only to colistin (Table 1). Although they are commensals, these isolates have the potential to become pathogens (Table 1). The results of the molecular approaches (transformation, conjugation and plasmid DNA hybridization experiments with a blaCTX-M-15 probe, which repeatedly failed; hybridization of the blaCTX-M-15 probe in all the isolates with a 280 kb XbaI PFGE DNA band absent from the plasmid
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Br S. Braenderup H9812 1 - 05E00174 2 - 12E226 3 - 11E851 4 - 12E587 5 - 11E604 6 - O104 7 - R363 8 - 12E937 9 - 12E276 10 - R261 11 - R208 12 - 10E0080 13 - R107 14 - R37 15 - R283
1135
blaCTX-M-15 in non-human German Escherichia coli and Salmonella
Congress of Clinical Microbiology and Infectious Diseases, Berlin, Germany, 2013 (Abstract 2740). We thank S. Schmoger, K. Thomas, S. Jahn, P. Trelka, S. Haby and W. Barownick for their technical support, the staff of the Department of Biological Safety (BfR), especially the NRL-E. coli staff, for surveillance and the analyses carried out during the EAEC-STEC O104 outbreak, B. Appel for his support and all the veterinary and public health laboratories for diagnostic and monitoring samples. Within the RESET Project, we thank: C. von Salviati, H. Laube A. Friese and U. Roesler (Free University Berlin, Germany) and K. Hille, J. Hering and Kreienbrock (University of Hannover, Germany) for epidemiological studies; and G. Brenner Michael and S. Schwarz for their scientific support. We thank E. Tietze (Robert Koch Institute), H. Hasman and R. Hendriksen (Danish Technical University, DTU, Denmark) and A. Carattoli (Istituto Superiore die Sanita, Italy) for providing control strains. We thank A. Battisti and P. de Pinto (Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Italy) for the Array Parser analysis.
Funding This work was supported by the Federal Institute for Risk Assessment, BfR (BfR-46-001; 46-002; 45-005) and the RESET Project (FKZ01Kl1013B and FKZ01Kl131B; BMBF, German Federal Ministry for Education and Research). E. G. was funded for a 3 month stay at the BfR by a fellowship from the Sociedad Espan˜ola de Enfermedades Infecciosas y Microbiologı´a Clı´nica (SEIMC).
Transparency declarations None to declare.
Supplementary data Table S1, Table S2 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 EFSA Panel on Biological Hazards (BIOHAZ). Scientific opinion on the public health risks of bacterial strains producing extended-spectrum b-lactamases and/or AmpC b-lactamases in food and food-producing animals. EFSA J 2011; 9: 2322. 2 D’Andrea MM, Arena F, Pallecchi L et al. CTX-M-type b-lactamases: a successful story of antibiotic resistance. Int J Med Microbiol 2013; 303: 305–17. 3 Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother 2009; 53: 2227– 38. 4 Ewers C, Bethe A, Semmler T et al. Extended-spectrum b-lactamaseproducing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect 2012; 18: 646– 55. 5 Pfeifer Y, Eller C, Leistner R et al. ESBL producer as human pathogens and the zoonotic reservoir. Hyg Med 2013; 38: 294–9. 6 Rodrı´guez I, Thomas K, van Essen A et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents 2014; 43: 553–7.
Acknowledgements
7 Rogers BA, Sidjabat HE, Paterson DL. Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother 2011; 66: 1– 14.
Part of this work was presented at the ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens, Aix-enProvence, France, 2012 (Abstract 112C) and the Twenty-third European
8 Price LB, Johnson JR, Aziz M et al. The epidemic of extended-spectrumb-lactamase-producing Escherichia coli ST131 is driven by a single highly pathogenic subclone, H30-Rx. MBio 2013; 4: e00377–13.
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profiles of the parental strains, Figure S1) indicated the presence of a chromosomally located blaCTX-M-15 gene in these isolates. As for the rest of the E. coli isolates included in the present study, the association of blaCTX-M-15 genes with ISEcp1 elements (located 48 bp upstream of the blaCTX-M-15 gene) was confirmed, but in six of the ST410 isolates the tnpA gene of this element was disrupted by the insertion of IS1 (Table 1). Only one of the ST410 isolates (R37) harboured an additional copy of the blaCTX-M-15 gene on an IncFA/FB/FII plasmid (110 kb, lacking blaOXA-1, blaTEM-1 and aac(6 ′ )-Ib-cr, but with the dfrA17-aadA5 integron). The adjacent ISEcp1 element may have driven the integration event of the blaCTX-M-15 gene into the chromosome by transposition or recombination in a progenitor strain of the ST410 isolates described. Although the chromosomal location of blaCTX-M-15 genes in different loci has previously been described,6,8 this is to our knowledge the first report of the clonal spread of a chromosomally encoded CTX-M-15 enzyme in livestock. Ongoing full sequencing analysis of these isolates (data not shown) may reveal the location of the gene in the chromosome and the genetic events implicated in the development of this clone. The increasing occurrence of ESBLs/AmpC in food-producing animals and food products observed during recent years in different countries is considered to be a public health threat.1,4,9,10 The possibility of the transmission of resistance genes and especially of ESBL genes via food was in fact highlighted by the severe German outbreak in 2011 caused by the EAEC-STEC O104:H4-B1-ST678 blaCTX-M-15 associated with the consumption of fenugreek sprouts.12,13,19 In that case, high virulence was combined with multiresistance traits that hampered treatment in the few cases in which it was suitable. In Germany the occurrence of multidrug-resistant CTX-M-15-producing E. coli isolates mainly originating from food-producing animals and foods is related to the spread of some clonal lineages such as ST678 (influenced by the German EHEC O104 outbreak in 2011), CC10 (ST167/ST617) and ST410 as well as IncI1 and multireplicon IncF plasmids. The ST410 clonal lineage harbours the blaCTX-M-15 gene integrated into the chromosome, promoting the maintenance of the gene in the livestock E. coli population. Although the clone O25:H4-B2-ST131 was not detected, most STs and serogroups observed are common in human E. coli, also being involved in clinical processes. An anthropogenic source for the recent increase in CTX-M-15producing E. coli from non-human specimens cannot be ruled out. Although Salmonella seems to play a less important role in the transmission of this gene (in our study only two isolates carried the gene, located on the IncHI2 and IncI1 plasmids), plasmid exchange does occur between different genera of Enterobacteriaceae. The role of a livestock reservoir for multidrugresistant bacteria and resistance mechanisms has been underlined in several studies.1,5 For all these reasons, the possibility of an exchange of isolates or plasmids between livestock and humans (i.e. via food products in one direction or contact contamination of farm workers in both directions) deserves continuous surveillance.
JAC
Fischer et al.
9 Wu G, Day MJ, Mafura MT et al. Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany. PLoS One 2013; 8: e75392.
15 Leverstein-van Hall MA, Dierikx CM, Cohen Stuart J et al. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 2011; 17: 873– 80.
10 Seiffert SN, Hilty M, Perreten V et al. Extended-spectrum cephalosporinresistant Gram-negative organisms in livestock: an emerging problem for human health? Drug Resist Update 2013; 16: 22–45.
16 Lo´pez-Cerero L, Egea P, Serrano L et al. Characterisation of clinical and food animal Escherichia coli isolates producing CTX-M-15 extendedspectrum b-lactamase belonging to ST410 phylogroup A. Int J Antimicrob Agents 2011; 37: 365–7. 17 Valenza G, Nickel S, Pfeifer Y et al. Extended-spectrum-b-lactamaseproducing Escherichia coli as intestinal colonizers in the German community. Antimicrob Agents Chemother 2014; 58: 1228– 30.
12 Beutin L, Martin A. Outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany causes a paradigm shift with regard to human pathogenicity of STEC strains. J Food Prot 2012; 75: 408–18.
18 Wieler LH, Semmler T, Eichhorn I et al. No evidence of the Shiga toxinproducing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC) found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area. Gut Pathog 2011; 3: 17.
13 Karch H, Denamur E, Dobrindt U et al. The enemy within us: lessons from the 2011 European Escherichia coli O104:H4 outbreak. EMBO Mol Med 2012; 4: 841–8.
19 Miko A, Delannoy S, Fach P et al. Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources. Int J Med Microbiol 2013; 303: 410–21.
14 Fischer J, Rodriguez I, Schmoger S et al. Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm. J Antimicrob Chemother 2012; 67: 1793– 5.
20 Schink A, Kadlec K, Schwarz S. Analysis of blaCTX-M-carrying plasmids from Escherichia coli isolates collected in the BfT-GermVet Study. Appl Environm Microbiol 2011; 77: 7142 –6.
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11 Rodrı´guez I, Barownick W, Helmuth R et al. Extended-spectrum b-lactamases and AmpC b-lactamases in ceftiofur-resistant Salmonella enterica isolates from food and livestock obtained in Germany during 2003– 07. J Antimicrob Chemother 2009; 64: 301– 9.
J Antimicrob Chemother doi:10.1093/jac/dku270
blaCTX-M-15-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany
1
Federal Institute for Risk Assessment, National Reference Laboratory for Antimicrobial Resistance, Department Biological Safety, Max-Dohrn Straße 8-10, D-10589 Berlin, Germany; 2Hospital Universitario Ramo´n y Cajal, Servicio de Microbiologı´a, Madrid, Spain; 3 Barcelona Centre for International Health Research (CRESIB), Hospital Clı´nic—Universidad de Barcelona, Barcelona, Spain; 4Robert Koch Institute, FG13 Nosocomial Pathogens and Antimicrobial Resistance, Wernigerode, Germany *Corresponding author. Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany. Tel: +49-30-18412-2082; Fax: +49-30-18412-2953; E-mail: [email protected]
Received 4 December 2013; returned 18 January 2014; revised 10 June 2014; accepted 21 June 2014 Objectives: The characterization of CTX-M-15 b-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. Methods: E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to thirdgeneration cephalosporins, originating from routine and monitoring submissions (2003 – 12) to the Federal Institute for Risk Assessment and different national targeted studies (2011–12), were examined for the presence of blaCTX-M-15 genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-15 were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. Results: Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-15 producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-15 genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. Conclusions: The prevalence of CTX-M-15-producing isolates from non-human sources in Germany is still low. The blaCTX-M-15 gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-15 gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-15-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance. Keywords: antimicrobial resistance, CTX-M, ESBLs, plasmids, chromosomal encoded
Introduction Worldwide the number of bacteria resistant to third- and fourthgeneration cephalosporins has increased (http://www.ecdc.europa. eu/en/activities/surveillance/EARS-Net).1 In most cases resistance is caused by the production of extended-spectrum b-lactamases (ESBLs) or AmpC-type b-lactamases. In Enterobacteriaceae, members of the CTX-M family are the predominant ESBLs.2 The b-lactamase blaCTX-M genes are in most cases located on transmissible genetic elements such as plasmids and transposable elements including insertion sequences (ISs) such as ISEcp1.3 The spread of
the blaCTX-M-15 gene, which encodes the most widely distributed CTX-M enzyme in Enterobacteriaceae of human origin,1,2,4 – 6 is mainly related to the rapid dissemination of the highly virulent extraintestinal pathogenic Escherichia coli clone O25:H4-B2-ST131 (implicated in human urinary tract and nosocomial infections).7 – 9 However, none of the ESBL-ST131-positive isolates from livestock or foods described so far has been a CTX-M-15 producer.1,4,9,10 In livestock CTX-M-1 is the most frequent ESBL in E. coli and Salmonella, and only sporadic occurrences of CTX-M-15producing E. coli in swine, cattle and poultry isolates have been described.1,4,9 – 11
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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Jennie Fischer1, Irene Rodrı´guez1,2, Beatrice Baumann1, Elisabeth Guiral1,3, Lothar Beutin1, Andreas Schroeter1, Annemarie Kaesbohrer1, Yvonne Pfeifer4, Reiner Helmuth1 and Beatriz Guerra1*
Fischer et al.
Materials and methods Bacterial isolates, b-lactam susceptibility testing and detection of ESBL/AmpC-encoding genes In total, 677 non-human E. coli (526) and Salmonella (151) isolates originating mainly from food and healthy food-producing animals (including 4 E. coli and 14 Salmonella isolates from companion and wild animals, feed, manure and a toy) were analysed. Of these isolates, 418 (270 E. coli and 148 Salmonella collected from January 2003 to July 2012 and submitted to the National Reference Laboratories NRL-AR, NRL-Salm and NRL-E. coli of the BfR) originated from routine diagnostic and national monitoring and surveillance programmes (i.e. commensal E. coli, verotoxin-producing E. coli and Salmonella; swine, cattle, turkey, poultry and their respective meats) and showed MIC values of cefotaxime or ceftazidime above the EUCAST epidemiological cut-offs (0.25 and 0.5 mg/L, respectively; http://www.eucast.org). The other 259 isolates (256 E. coli and three Salmonella) were collected using MacConkey agar supplemented with 1 mg/L cefotaxime as a selective medium in different targeted studies [66 swine, poultry and cattle farms; January 2011 and July 2012; Table S1 (available as Supplementary data at JAC Online)] within the national RESET Project (www.reset-verbund.de). All isolates were tested for their susceptibility to 17 b-lactams by the disc diffusion method (CLSI M2-A10) and for the presence of ESBL/ AmpC-encoding genes by PCR amplification/sequencing as previously described.6,11,14 For comparison, three human control strains were investigated.
Typing of CTX-M-15-producing isolates and characterization of antimicrobial resistance and virulence determinants Isolates positive for blaCTX-M-15 (23) were analysed by serotyping, XbaI PFGE (www.pulsenetinternational.org), multilocus sequence typing (MLST; http://mlst.warwick.ac.uk/) and phylogenetic grouping (E. coli) as previously described.6,11 The resistance phenotypes of the isolates were determined by testing their antimicrobial susceptibility to 14 antimicrobials by broth microdilution (CLSI M7-A8) (http://www.bfr.bund.de/cm/350/german-antimicrobialresistance-situation-in-the-food-chain-2009.pdf). The presence of additional resistance genes and integrons, as well as virulence genes, was screened using DNA microarrays (E. coli Genotyping Kit, Alere Technologies GmbH, Jena, Germany) following the manufacturer’s recommendations (http://alere-technologies.com/fileadmin/Media/Downloads/op/50202/ 05_16_04_0006_V05_Manual_E.coli.pdf) and/or PCR amplification/sequencing.6,11 The expression of the b-lactamase genes was tested by isoelectric focusing.6 Linkage of ISEcp1 with the blaCTX-M-15 genes was analysed by PCR amplification/sequencing.11 Plasmids were isolated by alkali lysis and transformed into E. coli DH10B Competent Cells.6,11 The plasmid content of the CTX-M-15-producing isolates and transformants (selected on MacConkey agar plates supplemented with 1 mg/L cefotaxime) was visualized by S1 PFGE.6,11 The location of the blaCTX-M-15 gene was tested by Southern blot
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hybridization of the S1, XbaI PFGE and Kado plasmid gels with a blaCTX-M-15 probe.6,11 Incompatibility (Inc) groups of plasmids were determined by PCR replicon typing (PBRT kit, Diatheva, Fano, Italy). IncI1 and IncF plasmids were analysed by plasmid MLST (pMLST; www.pubmlst.org/ plasmid/, accessed 18 July 2013). Conjugation experiments of selected blaCTX-M-15-carrying ST410 isolates (10 isolates) were performed by filter mating at 378C and room temperature.6,11
Results and discussion Among the 677 third-generation cephalosporin-resistant isolates analysed (526 E. coli and 151 Salmonella isolated mainly from food and healthy food-producing animals until July 2012), only 23 CTX-M-15-producing isolates (21 E. coli from livestock, food and a toy and 2 Salmonella from animals; 4.0% and 1.3%, respectively) were found (Table 1). The first CTX-M-15-producing E. coli isolated from livestock (Table 1) was collected in 2010, suggesting a recent emergence of this gene in the food-producing animal E. coli population in Germany. As described by other authors,1,4,9,10,15,16 also in bacteria from livestock and foods in Germany, the prevalence of blaCTX-M-15 is still low, in contrast to the high prevalence of E. coli with blaCTX-M-15 in humans in Germany.5,17 In our series, however, this prevalence varied among the studies in which isolates were collected, as shown in Table S1. To investigate whether the presence of CTX-M-15 producers was related to clonal spread or to the location of blaCTX-M-15 on mobile genetic elements, the molecular properties of the 21 E. coli and the two Salmonella isolates were analysed (Table 1 and Figure 1). MLST revealed that none of the E. coli isolates belonged to the previously mentioned E. coli O25:H4-B2-ST131, excluding the possible spread of this clone as a source for CTX-M-15-producing isolates in livestock and foods in Germany. On the other hand, three of the isolates were epidemiologically related to the German E. coli O104:H4-B1-ST678-blaCTX-M-15 outbreak strain and were recovered from an active surveillance of foods and patient’s households (Table 1 and Figure 1). After the outbreak in 2011, E. coli O104: H4-B1-ST678 isolates were rarely found in humans, and they had never previously been detected in food or animals.18,19 These isolates were highly pathogenic, characterized by the presence of several genes associated with enteroaggregative and enterohaemorrhagic E. coli (EHEC) strains as well as multidrug resistance.12,13,19 The isolates carried the blaCTX-M-15 gene on an 80 kb IncI1-pST31 plasmid (Table 1). IncI1 plasmids, in particular IncI1-pST31 and pST37 (http://pubmlst.org/; access date 28 June 2013; both plasmid types present in other isolates of the series), play an important role in the spread of blaCTX-M-15 in both humans and animals.3,10,15 A second detected clone comprised four E. coli isolates originating from turkey meat and swine (collected between January and May 2012) and ascribed to ST167/ST617, clonal complex CC10 (Table 1 and Figure 1). ESBL-producing E. coli isolates with these STs are commonly reported from humans but seldom from animals.4,9,10 The four isolates carried the blaCTX-M-15 genes on large multireplicon IncFIA/FIB/FII plasmids. These IncF plasmids carried additional genes [blaOXA-1, aac(6′ )-1b-cr or class 1 integrons; Table 1] characteristic of O25:H4-B2-ST131 IncF plasmids.3,7 Other E. coli blaCTX-M-15 isolates detected in the series also belonged to ST types (ST117, ST354, ST162 and ST196) commonly found among human E. coli isolates (http://mlst.ucc.ie/mlst/dbs/).
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In 2011 an enteroaggregative Shiga-toxin-producing E. coli (EAEC-STEC) O104:H4-B1-ST678 strain producing CTX-M-15 caused one of the severest foodborne outbreaks in Germany and Europe, affecting more than 3842 people.12,13 To look for a possible reservoir of this strain in animals or foods and to ascertain a potential relationship between the spread of CTX-M-15 producers in humans, livestock and food, we characterized all non-human CTX-M-15-producing E. coli and Salmonella isolates from the strain collections of the Federal Institute for Risk Assessment (BfR).
ALA3/ALA4 amplicon
Resistance Federal state Isolate no.
Species
05E00174f
E. coli
Source
phenotype/
(isolation date) resistance genotype
human
B (12/04/05)
(infection)
[AMP CTX CAZ]* CIP
Serotypea O25:H4
Phylogroupb B2
PFGE
MLST (clonal
pattern
complex)
E-X1
Inc Size
group
(ISEcp1 pMLST
ST131 (none) 110 kb FIA, FII F2:A1
sequence)c
Class 1 integrond
—
1700 bp/
NAL KAN STR SMX
Virulence gene(s)e iha, nfaE, prfB, sat
dfrA17-aadA5
TET TMP/ [blaCTX-M-15 blaOXA-1], aac(6 ′ )-Ib-cr, [aadA5, strA/B], [sul1, sul2], tet(A), dfrA17 12E00226
E. coli
meat, turkey
NRW (14/ 03/12)
[AMP CTX CAZ]* CIP
O1:Hr
D
E-X7
NAL SMX/
noneg
—
ST117 (none) 80 kb
I1
ST354
383 bph
—
lpfA
383 bph
—
lpfA, prfB, pic, vat, cba,
(CC354)
blaCTX-M-15, sul2 11E00851
E. coli
cattle
NRW (06/
(commensal)
06/11)
[AMP CTX CAZ]* CIP
ONT:HNT
D
E-X3
ST31
NAL STR SMX TET
cma, mcmA
TMP/[blaCTX-M-15, blaTEM-1], strA/B, sul2, tet(A), dfrA14 12E00587
E. coli
meat, calf
NRW (03/ 05/12)
[AMP CTX CAZ]* CHL
O55:HNT (fliCH10)
B1
E-X8
CIP NAL GEN KAN
ST162
85 kb
I1
ST37
383 bpi
—
lpfA, mchB, mchC, mchF
(CC469)
STR SMX TET TMP/ [blaCTX-M-15, blaTEM-1], catA1, aphA1, [aadA5, strA/B], sul2, tet(B), dfr17 11E00604
E. coli
cattle
NI (20/04/11)
(commensal)
[AMP CTX CAZ]* TET/
O8:H7
B1
E-X2
ST196 (none) 80 kb
I1
ST31
383 bph
—
f17-G, lpfA, cdtB, cnf1
O104:H4
B1
E-X6
ST678 (none) 80 kb
I1
ST31
383 bph
700 bp/dfrA7
stx2a, aggA, aap, aatA,
blaCTX-M-15 in non-human German Escherichia coli and Salmonella
Table 1. Phenotypic and molecular features of CTX-M-15-producing non-human enterobacterial isolates from Germany, 2005 –12
[blaCTX-M-15, blaTEM-1], tet(A)
O104 outbreak
E. coli
n ¼5 (NRZ-12-2027f, f
11E03787 ,
human (2,
ST (2), HE, NI
[AMP CTX CAZ]* STR
infection),
(2) (06-15/
SMX TET TMP/
aggR, pic, set1iha,
salmon,
06/11)
[blaCTX-M-15,
fyuA, irp2, terB, terD k
blaTEM-1], strA/B,
cucumber, toy
11E03789j,
[sul1, sul2],
11E03786j and
tet(A), dfrA7
11E03794j ) R363
E. coli
swine
BY (25/04/12)
(commensal)
[AMP CTX CAZ]* CIP
ONT:H10
A
E-X10
ST617 (CC10) 170 kb FII,
F31:A4:B1
NAL GEN SMX TET
FIA,
TMP/[blaCTX-M-15,
FIB
383 bph
1700 bp/
(astA)
dfrA17-aadA5
blaOXA-1], aac(6 ′ )-Ib-cr, aadA5, [sul1, sul2], tet(B), dfrA17
Continued
JAC
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ALA3/ALA4 amplicon
Resistance Federal state Isolate no. 12E00937
Species E. coli
Source
phenotype/
(isolation date) resistance genotype
meat, turkey
NRW (23/ 05/12)
[AMP CTX CAZ]* CIP
Serotypea ONT:HNM
Phylogroupb A
PFGE
MLST (clonal
pattern
complex)
E-X9
Inc Size
group
(ISEcp1 pMLST F31:A4:B37l
ST167 (CC10) 140 kb FII,
NAL STR SMX TET/
FIA,
[blaCTX-M-15,
FIB
sequence)c
Class 1 integrond
383 bph
—
Virulence gene(s)e Iha, sfaS, (espA_C_ rodentium), senB
blaOXA-1], aac(6 ′ )-Ib-cr, strA/B, sul2, tet(A) 12E00276
E. coli
meat, turkey
NRW (04/ 04/12)
[AMP CTX CAZ]* CIP
ONT:HNM
A
E-X5b
ST167 (CC10) 170 kb FII,
F31:A4:B1
NAL STR SMX TET
FIA,
TMP/[blaCTX-M-15,
FIB
383 bph
1700 bp/ dfrA17-aadA5
blaOXA-1], aac(6 ′ )-Ib-cr, [aadA5, strA/B], tet(A), dfrA17 R261
E. coli
swine
BB (09/01/12)
(commensal)
[AMP CTX CAZ]* CIP
ONT:H9
A
E-X5a
ST167 (CC10) 145 kb FII,
F31:A4:B1
NAL KAN STR SMX
FIA,
TET/[blaCTX-M-15,
FIB
383 bph
—
—
lpfA, cma
blaOXA-1], aac(6 ′ )-Ib-cr, strA/B, sul2, tet(A) R208
E. coli
swine
NRW (29/
(commensal)
06/11)
[AMP CTX CAZ]* CIP
O8:H9
A
E-X4d
ST410 (CC23) noneg
—
382 bph
—
Or:H9
A
E-X4a
ST410 (CC23) noneg
—
382 bph
1600 bp/dfrA1-
NAL TET/ blaCTX-M-15, [tet(A), tet(B)]
10E00080
E. coli
cattle
NI (15/02/10)
(commensal)
[AMP CTX CAZ]* CHL CIP NAL STR SMX
aadA1;
TET TMP/
1900 bp/
[blaCTX-M-15,
drfA12-aadA2
cma
blaTEM-1], catA1, [aadA1, aadA2, strA/B], [sul1, sul3], tet(B), [dfrA1, drfA12] R107
E. coli
cattle
NRW (29/
(commensal)
06/11)
[AMP CTX CAZ]* CIP
O8:HNT (fliCH9)
A
E-X4b
ST410 (CC23) noneg
382 bph
—
NAL STR SMX TET
1900 bp/
lpfA, cma
dfrA12-aadA2
TMP/blaCTX-M-15, aadA2, sul1, tet(A), dfrA12 E. coli
swine (commensal)
ST410 (CC23) 110 kbg FII,
NRW (24/
[AMP CTX CAZ]* CHL
05/11)
CIP NAL STR SMX
FIA,
TMP/[blaCTX-M-15,
FIB
O20:H9
A
E-X4e
F31:A4:B1
382 bph
1700 bp/ dfrA17-aadA5
blaOXA-1], aac(6 ′ )-Ib-cr, [aadA5, strA/B], [sul1, sul2], dfrA17
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R37
(lpfA, sfaS), senB
Fischer et al.
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Table 1. Continued
E. coli
swine (3), poultry NRW (n ¼4, 14/ [AMP CTX CAZ]* CHL
ONT:HNM (fliC
R61a, R283,
(2), cattle (1)
06/12-04/
CIP NAL FFN GEN
H21) (5),
R341 and R345)
(commensals)
07/11), BY
KAN STR SMX TET
ONT:H21
(23/04/12),
TMP/blaCTX-M-15,
TH (17/
catA1, floR,
08/11)
[aadA5, strA/B]
A
E-X4c
ST410 (CC23) noneg
1172 bpm
—
1700 bp/ dfrA17-aadA5
f17-G, (f17-A)+ (lpfA, espB_O26, espA_C_rodentium; espP)
aadA5, sul1, tet(A), dfrA17 05-01901
Salmonella horse (infection)
HE (27/04/05)
[AMP CTX CAZ]* STR SMX TET TMP/
Salmonella
S-X1
not done
90 kb
I1
ST31
383 bph
800 bp/aadB; 1200 bp/
Typhimurium
[blaCTX-M-15,
blaPSE-1
blaTEM-1, blaPSE-1], aadB, sul1, tet(G) 09-1454
Salmonella swine
HE (17/04/09)
(commensal)
[AMP CTX CAZ]* CHL CIP GEN STR SMX
Salmonella
S-X2
not done
320 kb HI2
not done
383 bph
1000 bp/aadA1
Bredeney
TET TMP/ [blaCTX-M-15, blaTEM-1, blaOXA-1], aac(6 ′ )-Ib-cr, aadA1, strA, sul2, tet(A), dfrA1-like
Abbreviations. Antimicrobials: AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; FFN, florfenicol; KAN, kanamycin; NAL, nalidixic acid; SMX, sulfamethoxazole; STR, streptomycin; TET, tetracycline; TMP, trimethoprim. Regions: B, Berlin; BY, Bavaria; HE, Hesse; NI, Lower Saxony; NRW, North Rhine-Westphalia; ST, Saxony-Anhalt. Others: r, rough; NM, non-motile; NT, not typeable. MICs were determined using CLSI methodology (M07-A8). Evaluation of the results was performed according to the European Community Decision 2007/407/EC (http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2007:153:0026:0029:EN:PDF) using the EUCAST epidemiological cut-off values (www.eucast.org). All isolates were susceptible to colistin. [AMP CTX CAZ]*: MIC phenotype. These isolates showed microbiological resistance to the penicillins and first- to third-generation cephalosporins and monobactams tested, and susceptibility to cephamycins and carbapenems, as obtained by the disc diffusion method following the EUCAST guidelines and cut-off values (www.eucast.org) as previously described.11 a Serotyping of E. coli, performed as described in Beutin et al. 12 Serotyping of the Salmonella isolates performed following the White– Kauffmann –Le Minor scheme (www.pasteur.fr). b Performed as described by Rodrı´guez et al.6 c PCR amplicons obtained using the ISEcp1-related primer ALA3 and a modified ALA4.6 d Approximate size of the amplicon obtained using the 5′ CS-3′ CS primers/gene contained in the variable region.11 e Virulence genes were analysed with the E. coli Genotyping Kit (Alere Microarray). Genes tested and row results obtained are shown in Table S2. Genes in brackets were recognized only with an ambiguous signal in the array. f Reference strain (control). Strain no. 05E00174, from a urinary tract infection received by the NRL-E. coli, represented the E. coli clone O25:H4-B2-ST131-blaCTX-M-15. Strains EHEC O104 NRZ-12-2027, kindly provided by Dr Tietze (Robert Koch Institute), and 11E03787 (received by the NRL-E. coli) represented the E. coli O104:H4-B1-ST678-blaCTX-M-15 and were isolated from patients during the German outbreak in 2011. g Chromosomally located blaCTX-M-15 gene. h Identical to HF549093.1 sequence. i Sequence identical to the one with accession number GQ385309.1. j Isolates related to the German EHEC O104:H4-ST678 outbreak: salmon recovered from a catering service prepared by an intoxicated patient (secondary outbreak), cucumber recovered from the garbage of an intoxicated family, toy recovered from a patient household. k Characterized by Miko et al.19 l Differs from IncFIB 1 in only one nucleotide. m The tnpA gene is disrupted by the insertion of IS1 (HG798331).
blaCTX-M-15 in non-human German Escherichia coli and Salmonella
n ¼ 6 (R54, R56,
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Fischer et al.
(a) kb
CC10
ST678 Br 1
2
3
4
5
6 Br
7
8
ST410
9 10 11 12 13 14 15 Br
452.7
244.4 138.9
33.3
ST410
(b) kb
Br 1
2
3
4
5
6
ST678 7
8
9 10 Br 11 12 13 14 15 Br
1135
452.7
244.4
*
138.9
33.3
Br S. Braenderup H9812 1 - R208 2 - 10E0080 3 - R107 4 - R37 5 - R283 6 - R54 7 - R56 8 - R61 9 - R345 10 - R341 11 - O104 12 - 11E3786 13 - 11E3787 14 - 11E3789 15 - 11E3794
*
Figure 1. XbaI PFGE patterns from blaCTX-M-15-positive E. coli. (a) All different XbaI PFGE patterns. CC10 includes isolates of ST167 (lanes 8– 10) and 617 (lane 7). (b) XbaI PFGE patterns from E. coli isolates belonging to the A-ST410 clone (*280 kb band, which hybridized with a blaCTX-M-15 probe) or B1-ST678 clone.
One of the most interesting findings of the present work was the detection of the widespread E. coli ST410 phylogroup A clonal line. E. coli ST410 are frequently isolated from humans and animal sources and have also been recovered from human clinical samples.4,10,16,20 The isolates from the present study ascribed to this line (10) were collected from swine, cattle and poultry farms in four German federal states from February 2010 until July 2012 [Table 1, Figure 1 and Figure S1 (available as Supplementary data
6 of 8
at JAC Online)]. These isolates were resistant to 13 of the 14 antimicrobials tested, retaining susceptibility only to colistin (Table 1). Although they are commensals, these isolates have the potential to become pathogens (Table 1). The results of the molecular approaches (transformation, conjugation and plasmid DNA hybridization experiments with a blaCTX-M-15 probe, which repeatedly failed; hybridization of the blaCTX-M-15 probe in all the isolates with a 280 kb XbaI PFGE DNA band absent from the plasmid
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Br S. Braenderup H9812 1 - 05E00174 2 - 12E226 3 - 11E851 4 - 12E587 5 - 11E604 6 - O104 7 - R363 8 - 12E937 9 - 12E276 10 - R261 11 - R208 12 - 10E0080 13 - R107 14 - R37 15 - R283
1135
blaCTX-M-15 in non-human German Escherichia coli and Salmonella
Congress of Clinical Microbiology and Infectious Diseases, Berlin, Germany, 2013 (Abstract 2740). We thank S. Schmoger, K. Thomas, S. Jahn, P. Trelka, S. Haby and W. Barownick for their technical support, the staff of the Department of Biological Safety (BfR), especially the NRL-E. coli staff, for surveillance and the analyses carried out during the EAEC-STEC O104 outbreak, B. Appel for his support and all the veterinary and public health laboratories for diagnostic and monitoring samples. Within the RESET Project, we thank: C. von Salviati, H. Laube A. Friese and U. Roesler (Free University Berlin, Germany) and K. Hille, J. Hering and Kreienbrock (University of Hannover, Germany) for epidemiological studies; and G. Brenner Michael and S. Schwarz for their scientific support. We thank E. Tietze (Robert Koch Institute), H. Hasman and R. Hendriksen (Danish Technical University, DTU, Denmark) and A. Carattoli (Istituto Superiore die Sanita, Italy) for providing control strains. We thank A. Battisti and P. de Pinto (Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Italy) for the Array Parser analysis.
Funding This work was supported by the Federal Institute for Risk Assessment, BfR (BfR-46-001; 46-002; 45-005) and the RESET Project (FKZ01Kl1013B and FKZ01Kl131B; BMBF, German Federal Ministry for Education and Research). E. G. was funded for a 3 month stay at the BfR by a fellowship from the Sociedad Espan˜ola de Enfermedades Infecciosas y Microbiologı´a Clı´nica (SEIMC).
Transparency declarations None to declare.
Supplementary data Table S1, Table S2 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 EFSA Panel on Biological Hazards (BIOHAZ). Scientific opinion on the public health risks of bacterial strains producing extended-spectrum b-lactamases and/or AmpC b-lactamases in food and food-producing animals. EFSA J 2011; 9: 2322. 2 D’Andrea MM, Arena F, Pallecchi L et al. CTX-M-type b-lactamases: a successful story of antibiotic resistance. Int J Med Microbiol 2013; 303: 305–17. 3 Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother 2009; 53: 2227– 38. 4 Ewers C, Bethe A, Semmler T et al. Extended-spectrum b-lactamaseproducing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect 2012; 18: 646– 55. 5 Pfeifer Y, Eller C, Leistner R et al. ESBL producer as human pathogens and the zoonotic reservoir. Hyg Med 2013; 38: 294–9. 6 Rodrı´guez I, Thomas K, van Essen A et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents 2014; 43: 553–7.
Acknowledgements
7 Rogers BA, Sidjabat HE, Paterson DL. Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother 2011; 66: 1– 14.
Part of this work was presented at the ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens, Aix-enProvence, France, 2012 (Abstract 112C) and the Twenty-third European
8 Price LB, Johnson JR, Aziz M et al. The epidemic of extended-spectrumb-lactamase-producing Escherichia coli ST131 is driven by a single highly pathogenic subclone, H30-Rx. MBio 2013; 4: e00377–13.
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profiles of the parental strains, Figure S1) indicated the presence of a chromosomally located blaCTX-M-15 gene in these isolates. As for the rest of the E. coli isolates included in the present study, the association of blaCTX-M-15 genes with ISEcp1 elements (located 48 bp upstream of the blaCTX-M-15 gene) was confirmed, but in six of the ST410 isolates the tnpA gene of this element was disrupted by the insertion of IS1 (Table 1). Only one of the ST410 isolates (R37) harboured an additional copy of the blaCTX-M-15 gene on an IncFA/FB/FII plasmid (110 kb, lacking blaOXA-1, blaTEM-1 and aac(6 ′ )-Ib-cr, but with the dfrA17-aadA5 integron). The adjacent ISEcp1 element may have driven the integration event of the blaCTX-M-15 gene into the chromosome by transposition or recombination in a progenitor strain of the ST410 isolates described. Although the chromosomal location of blaCTX-M-15 genes in different loci has previously been described,6,8 this is to our knowledge the first report of the clonal spread of a chromosomally encoded CTX-M-15 enzyme in livestock. Ongoing full sequencing analysis of these isolates (data not shown) may reveal the location of the gene in the chromosome and the genetic events implicated in the development of this clone. The increasing occurrence of ESBLs/AmpC in food-producing animals and food products observed during recent years in different countries is considered to be a public health threat.1,4,9,10 The possibility of the transmission of resistance genes and especially of ESBL genes via food was in fact highlighted by the severe German outbreak in 2011 caused by the EAEC-STEC O104:H4-B1-ST678 blaCTX-M-15 associated with the consumption of fenugreek sprouts.12,13,19 In that case, high virulence was combined with multiresistance traits that hampered treatment in the few cases in which it was suitable. In Germany the occurrence of multidrug-resistant CTX-M-15-producing E. coli isolates mainly originating from food-producing animals and foods is related to the spread of some clonal lineages such as ST678 (influenced by the German EHEC O104 outbreak in 2011), CC10 (ST167/ST617) and ST410 as well as IncI1 and multireplicon IncF plasmids. The ST410 clonal lineage harbours the blaCTX-M-15 gene integrated into the chromosome, promoting the maintenance of the gene in the livestock E. coli population. Although the clone O25:H4-B2-ST131 was not detected, most STs and serogroups observed are common in human E. coli, also being involved in clinical processes. An anthropogenic source for the recent increase in CTX-M-15producing E. coli from non-human specimens cannot be ruled out. Although Salmonella seems to play a less important role in the transmission of this gene (in our study only two isolates carried the gene, located on the IncHI2 and IncI1 plasmids), plasmid exchange does occur between different genera of Enterobacteriaceae. The role of a livestock reservoir for multidrugresistant bacteria and resistance mechanisms has been underlined in several studies.1,5 For all these reasons, the possibility of an exchange of isolates or plasmids between livestock and humans (i.e. via food products in one direction or contact contamination of farm workers in both directions) deserves continuous surveillance.
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Fischer et al.
9 Wu G, Day MJ, Mafura MT et al. Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany. PLoS One 2013; 8: e75392.
15 Leverstein-van Hall MA, Dierikx CM, Cohen Stuart J et al. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 2011; 17: 873– 80.
10 Seiffert SN, Hilty M, Perreten V et al. Extended-spectrum cephalosporinresistant Gram-negative organisms in livestock: an emerging problem for human health? Drug Resist Update 2013; 16: 22–45.
16 Lo´pez-Cerero L, Egea P, Serrano L et al. Characterisation of clinical and food animal Escherichia coli isolates producing CTX-M-15 extendedspectrum b-lactamase belonging to ST410 phylogroup A. Int J Antimicrob Agents 2011; 37: 365–7. 17 Valenza G, Nickel S, Pfeifer Y et al. Extended-spectrum-b-lactamaseproducing Escherichia coli as intestinal colonizers in the German community. Antimicrob Agents Chemother 2014; 58: 1228– 30.
12 Beutin L, Martin A. Outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany causes a paradigm shift with regard to human pathogenicity of STEC strains. J Food Prot 2012; 75: 408–18.
18 Wieler LH, Semmler T, Eichhorn I et al. No evidence of the Shiga toxinproducing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC) found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area. Gut Pathog 2011; 3: 17.
13 Karch H, Denamur E, Dobrindt U et al. The enemy within us: lessons from the 2011 European Escherichia coli O104:H4 outbreak. EMBO Mol Med 2012; 4: 841–8.
19 Miko A, Delannoy S, Fach P et al. Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources. Int J Med Microbiol 2013; 303: 410–21.
14 Fischer J, Rodriguez I, Schmoger S et al. Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm. J Antimicrob Chemother 2012; 67: 1793– 5.
20 Schink A, Kadlec K, Schwarz S. Analysis of blaCTX-M-carrying plasmids from Escherichia coli isolates collected in the BfT-GermVet Study. Appl Environm Microbiol 2011; 77: 7142 –6.
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11 Rodrı´guez I, Barownick W, Helmuth R et al. Extended-spectrum b-lactamases and AmpC b-lactamases in ceftiofur-resistant Salmonella enterica isolates from food and livestock obtained in Germany during 2003– 07. J Antimicrob Chemother 2009; 64: 301– 9.
