friedrich-loeffler-institut (fli) institute of farm animal

Friedrich-Loeffler-Institut (FLI)

Institute of Farm Animal Genetics

Neustadt-Mariensee, Germany

Studies on the prevalence, distribution and organization of

extended-spectrum �-lactamase genes and transferable

(fluoro)quinolone resistance genes among Enterobacteriaceae from

defined disease conditions of companion and farm animals

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by

Anne-Kathrin Schink

from Hannover

Hannover 2012

Supervisor: Prof. Dr. Stefan Schwarz

Supervision Group: Prof. Dr. Stefan Schwarz

Prof. Dr. Günter Klein

Prof. Dr. Peter Heisig

1st Evaluation: Prof. Dr. Stefan Schwarz,

Friedrich-Loeffler-Institut (FLI)

Institute of Farm Animal Genetics, Neustadt-Mariensee

Prof. Dr. Günter Klein,

Institute of Food Quality and Food Safety, University of

Veterinary Medicine Hannover, Hannover

Prof. Dr. Peter Heisig,

Pharmaceutical Biology and Microbiology, Department of

Chemistry, University of Hamburg, Hamburg

2nd Evaluation: Prof. Dr. Dik Mevius,

Faculty of Veterinary Medicine, Department of Infectious

Diseases and Immunology, Utrecht University, Utrecht,

The Netherlands

Central Veterinary Institute of Wageningen, Lelystad,

The Netherlands

Date of final exam: 10 May 2012

Sponsorship: Anne-Kathrin Schink was supported by a scholarship of

the H. Wilhelm Schaumann foundation.

For my family and friends,

even though they think

my work is just bla, bla.

Parts of the thesis have already been published:

Schink, A.-K., Kadlec, K., & Schwarz, S. (2011). Analysis of blaCTX-M-carrying

plasmids from Escherichia coli isolates collected in the BfT-GermVet study. Appl.

Environ. Microbiol.,77, 7142-7146.

Schink, A.-K., Kadlec, K., & Schwarz, S. (2012). Detection of qnr genes among

Escherichia coli isolates of animal origin and complete sequence of the conjugative

qnrB19-carrying plasmid pQNR2078. J. Antimicrob. Chemother., 67, 1099-1102.

Further aspects have been presented at national or international conferences

as oral presentations or as posters:

Schink, A.-K., Kadlec, K., & Schwarz, S. (2009). Analysis of a blaCTX-M-1-carrying

plasmid from a canine Escherichia coli isolate collected in the BfT-GermVet study.

Proceedings of the 61th Jahrestagung der Deutschen Gesellschaft für Hygiene und

Mikrobiologie e.V. (DGHM), Göttingen, published in International Journal of Medical

Microbiology, poster PRP02, 299S1 (Suppl. 46), 76.

Schink, A.-K., Kadlec, K., & Schwarz, S. (2010). Analysis of blaCTX-M-1-carrying

plasmids from Escherichia coli isolates collected in the BfT-GermVet study in

Germany. Proceedings of the 2nd ASM Conference on Antimicrobial Resistance in

Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the

Environment, Toronto, Canada, oral presentation S2:6, 19.


plasmids from Escherichia coli isolates collected in the BfT-GermVet study in

Germany. Proceedings of the 2nd ASM Conference on Antimicrobial Resistance in

Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the

Environment, Toronto, Canada, poster A191, 117.

Schink, A.-K., Kadlec, K., & Schwarz, S. (2010). Comparative analysis of the

plasmid-borne blaCTX-M-1 regions of Escherichia coli from different animal sources.

Proceedings of the Tagung der Deutschen Veterinärmedizinischen Gesellschaft

(DVG), division „Bakteriologie und Mykologie”, Jena, oral presentation V29, 29.

Schwarz, S., Schink, A.-K., & Kadlec, K. (2010). Susceptibility to pradofloxacin

among various bacterial pathogens from dogs and cats. Proceedings of the Tagung

der Deutschen Veterinärmedizinischen Gesellschaft (DVG), division „Bakteriologie

und Mykologie”, Jena, poster P88, 124 .


plasmids from Escherichia coli isolates collected in the BfT-GermVet study.

Proceedings of the 462nd WE Heraeus Seminar, Jacobs University Bremen, Bremen,

poster (http://faculty.jacobs-university.de/mwinterhalter/heraeus transporters_2010/

poster/Anne-Kathrin_Schink.pdf –last accessed 15 March 2012).

Schink, A.-K., Kadlec, K., & Schwarz, S. (2010). Detection of qnrB19 genes among

Escherichia coli isolates collected in the BfT-GermVet study. Proceedings of the 50th

Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) of the

American Society for Microbiology (ASM), Boston, U. S. A., poster C2-1467.

Schink, A.-K., Kadlec, K., & Schwarz, S. (2011). Analysis of qnrB19-carrying

plasmids from equine Escherichia coli isolates. Proceedings of the 4th Symposium

on Antimicrobial Resistance in Animals and the Environment (ARAE), Tours, France,

poster P28, 113.

Schwarz, S., Schink, A.-K., Schiwek, M., & Kadlec, K. (2011). Susceptibility to

pradofloxacin among bacterial pathogens from dogs and cats. Proceedings of the 4th

Symposium on Antimicrobial Resistance in Animals and the Environment (ARAE),

Tours, France, poster P34, 118.

Contents Chapter 1 Introduction

9

Chapter 2 Analysis of blaCTX-M-carrying plasmids from Escherichia coli

isolates collected in the BfT-GermVet study

13

Chapter 3 Detection of qnr genes among Escherichia coli isolates of

animal origin and complete sequence of the conjugative

qnrB19-carrying plasmid pQNR2078

15

Chapter 4 Discussion 17

1. Occurrence of blaCTX-M genes in E. coli from Germany and

in other countries

18

2. The genetic environment of blaCTX-M genes 19

3. Occurrence of the qnrB19 gene in Germany and in other

countries

25

4. The genetic environment of the qnrB19 gene 26

5. Plasmids in Enterobacteriaceae 31

6. Animal reservoirs? 33

7. Concluding remarks

35

Chapter 5 Summary

37

Chapter 6 Zusammenfassung

41

References

45

Acknowledgements 57

- 9 -

Chapter 1

Introduction

Chapter 1 Introduction

- 10 -

Introduction

Antimicrobial agents are indispensable for the control of bacterial infections. Among

the diverse classes of antimicrobial agents, �-lactam antibiotics and nowadays

fluoroquinolones, which were considered as antibiotics of last resort, were frequently

used in human medicine in Germany (Kern & Nink, 2011; de With et al., 2011). In

veterinary medicine, little information about the application of antimicrobial agents is

currently available (Schneidereit, 2011). Instead, sales figures of active

pharmaceutical ingredients have been compiled for the year 2005 by the Federation

of Animal Health (Bundesverband für Tiergesundheit BfT) (Schneidereit, 2008).

Treatment failure is very often due to acquired resistance determinants, which limit

therapeutic options considerably.

During the last years the occurrence of extended-spectrum �-lactamases (ESBLs)

and plasmid-mediated quinolone resistances (PMQR) within the family

Enterobacteriaceae has gained particular attention. Members of this family can be

harmless colonisers of the gut, but some of them can cause severe gastrointestinal

and even extraintestinal infections in both humans and animals.

ESBLs have been first described in the early 1980s and confer resistance to �-

lactam antibiotics such as penicillins, cephalosporins and monobactams. The

corresponding genes have developed by point mutations from known narrow-

spectrum �-lactamase genes, namely blaSHV and blaTEM. The amino acid exchanges

which resulted from the point mutations led to an expansion of the hydrolysing

activity of these enzymes from penicillins to cephalosporins and monobactams. In

1989, a novel ESBL gene has been detected in an E. coli isolate from Germany,

which has been genetically unrelated to any known �-lactamase gene and

designated blaCTX-M-1 (Bauernfeind et al., 1990). According to Livermoore et al.

(2007), CTX-M ESBLs are the predominant ESBL type in Europe. Nowadays, 122

CTX-M ESBLs have been deposited in the Lahey database (http://www.lahey.org/

studies/webt.htm) and represent five distinct clusters based on their amino acid

sequence homology.

Introduction Chapter 1

- 11 -

The first PMQR gene, qnrA, has been described in 1998 (Martínez-Martínez et al.,

1998). Qnr proteins protect the DNA-gyrase-complex, the target of quinolones and

fluoroquinolones, and thus mediate resistance to quinolones and decreased

susceptibility to fluoroquinolones. More qnr genes, qnrB, qnrC, qnrD and qnrS, and

subtypes thereof have been identified (Jacoby et al., 2006; Wang et al., 2009;

Cavaco et al., 2009; Hata et al., 2005). Besides qnr genes the gene aac(6’)-Ib-cr,

coding for an aminoglycoside acetyltransferase, has been detected, which confers

resistance to kanamycin and decreased susceptibility to ciprofloxacin and norfloxacin

by acetylating their piperazinyl substituent (Robicsek et al., 2006). Two plasmid-

encoded efflux pumps, QepA1 and QepA2 (Yamane et al., 2007; Cattoir et al.,

2008b), have also been reported.

ESBL genes and PMQRs have been described to be colocated either on the same

plasmid or on different plasmids within the same isolate (Richter et al., 2010; Dionisi

et al., 2009; Müller et al., 2011; Woodford et al., 2009; Dolejska et al., 2011; Kirchner

et al., 2011; Yao et al., 2011).

The information about ESBLs and PMQRs in E. coli from diseased animals in

Germany is scarce. Thus, the aims of this project were

(i) to determine the presence of ESBL and PMQR genes in E. coli isolates from

defined disease conditions of companion and farm animals,

(ii) to gain insight into the localisation and organisation of the genetic

environment of the ESBL and PMQR genes, and

(iii) to obtain information about the transferability of the ESBL and PMQR genes.

- 12 -

- 13 -

Chapter 2

Analysis of blaCTX-M-carrying plasmids from

Escherichia coli isolates collected in the

BfT-GermVet study

Anne-Kathrin Schink, Kristina Kadlec and Stefan Schwarz

(2011), Appl. Environ. Microbiol., 77, 7142-7146

Chapter 2 Analysis of blaCTX-M gene regions

- 14 -

The extent of contribution from Anne-Kathrin Schink to the article is evaluated

according to the following scale:

A. has contributed to collaboration (0-33%).

B. has contributed significantly (34-66%).

C. has essentially performed this study independently (67-100%).

1. Design of the project including design of individual experiments B

2. Performing of the experimental part of the study C

3. Analysis of experiments C

4. Presentation and discussion of the study in article form C

- 15 -

Chapter 3

Detection of qnr genes among Escherichia coli isolates of

animal origin and complete sequence of the conjugative

qnrB19-carrying plasmid pQNR2078

Anne-Kathrin Schink, Kristina Kadlec and Stefan Schwarz

(2012), J. Antimicrob. Chemother., 67, 1099-1102

Chapter 3 qnrB19 in animal E. coli isolates

- 16 -

The extent of contribution from Anne-Kathrin Schink to the article is evaluated

according to the following scale:

A. has contributed to collaboration (0-33%).

B. has contributed significantly (34-66%).

C. has essentially performed this study independently (67-100%).

1. Design of the project including design of individual experiments C

2. Performing of the experimental part of the study C

3. Analysis of experiments C

4. Presentation and discussion of the study in article form C

- 17 -

Chapter 4

Discussion

Chapter 4 Discussion

- 18 -

Discussion

1. Occurrence of blaCTX-M genes in E. coli from Germany and other countries

The ESBLs corresponding to the three blaCTX-M genes found in this study belonged

to the CTX-M-1 group. ESBLs of this group are commonly found in E. coli isolates in

Europe and represent the predominant ESBL group in some other countries (Coque

et al., 2008; Livermore et al., 2007). In Germany, ESBL-producing E. coli isolates

with blaCTX-M-1 and blaCTX-M-15 genes of human and animal origin have been described

(Cullik et al., 2010; Ewers et al., 2010; Schmitt et al., 2007).

The gene blaCTX-M-15 has been described in isolates of the worldwide distributed E.

coli type O25:H4-ST131 (Nicolas-Chanoine et al., 2008; Woodford et al., 2011). In

Germany, CTX-M-15 ESBL-producing E. coli O25b-ST131 from dogs have been

found (Ewers et al., 2010). The E. coli ST131 clonal group as well as E. coli ST410

have shown an extended host spectrum genotype (EHSG) by their presence in

humans and animals (Wieler et al., 2011). According to the MLST database

(http://mlst.ucc.ie/mlst/dbs/Ecoli, last accessed 15 March 2012), E. coli ST410

isolates have been reported in human and bovine isolates from Canada and in

human isolates from Brazil, Ghana, England and Spain. E. coli ST410 isolates have

been further identified among CTX-M-15 ESBL-producing human isolates from the

U.S.A. and from Brazil (Sidjabat et al., 2009; Peirano et al., 2011) as well as in

Spanish isolates from humans and turkey meat (López-Cerero et al., 2011).

Therefore, E. coli ST410 isolates seem to be widely distributed among humans and

animals. The blaCTX-M-15-positive E. coli isolate 913 from a dog suffering from urinary

tract infection, identified in the present PhD project, also belonged to MLST type

ST410 and is the first of these isolates detected in companion animals.

The blaCTX-M-1 ESBL-producing E. coli isolates belonged to the novel MLST types

ST1153 and ST1576. However, after we firstly described ST1153 one more porcine

pathogenic E. coli ST1153 isolate from Germany has been deposited in the MLST-

database (http://mlst.ucc.ie/mlst/dbs/Ecoli, last accessed 15 March 2012), indicating

Discussion Chapter 4

- 19 -

that the finding of this type of E. coli has not been an accidental observation.

Nevertheless, blaCTX-M-1-carrying plasmids have been described in a multitude of

E. coli sequence types (Ben Sallem et al., 2011; Bonnedahl et al., 2009; Cullik et al.,

2010; Leverstein-Van Hall et al., 2011; Oteo et al., 2009) which is a hint towards a

horizontal rather than a clonal spread.

2. The genetic environment of blaCTX-M genes

The blaCTX-M-15 gene was located on an IncF plasmid (pCTX913) of ca. 50 kb in

size and the two blaCTX-M-1 genes on ca. 50 kb IncN plasmids (pCTX168 and

pCTX246). The analysis of the immediately flanking regions up- and downstream of

the ESBL genes on pCTX913, pCTX168 and pCTX246 revealed at least a fragment

of the insertion sequence ISEcp1 48 bp or 80 bp upstream and downstream the

terminal part of orf477. The same genetic environment has been described for these

genes before (Eckert et al., 2006; Lartigue et al., 2004). Poirel et al. (2005) proved

that ISEcp1B is able to mobilize blaCTX-M genes in a one-ended transposition process,

which in these cases might have included the ESBL genes and the terminal part of

orf477. The genetic environment of blaCTX-M-15 and blaCTX-M-1 is depicted in Figures 1

and 2 and the plasmids mentioned in the following text are listed in Table 1.

In clinical K. pneumoniae isolates from Nigeria, plasmids larger than 58 kb

carrying blaCTX-M-15 with the same immediately flanking 193 bp up- and 212 bp

downstream have been identified (Soge et al., 2006). Furthermore, the downstream

region of the gene blaCTX-M-15 on pCTX913 with the �orf477-�tnpA structure has been

described in a wide variety of plasmids and in conjunction with different bacterial

hosts. The 92,353 bp IncFII plasmid pC15-1a from a human clinical E. coli isolate

obtained in 2002 in Canada (Boyd et al., 2004) and pYC-5b, a ca. 50 kb plasmid

harboured by a human clinical E. coli isolate from Cameroon (Gangoue-Pieboji et al.,

2005), showed the same blaCTX-M-15 downstream region as in pCTX913. In E. coli

isolates of the international clone O25:H4-ST131 from the UK, different plasmids

have been identified and the plasmids pEK499 (117,536 bp in size and positive for


- 20 -

replicons FII and FIA) and pEK516 (64,471 bp in size and IncFII positive) showed the

same immediate flanking regions (Woodford et al., 2009). In a Belgian study four

plasmids, showing the same downstream region, were sequenced. Three were

obtained from human E. coli isolates and the fourth E. coli was of equine origin. The

plasmid pEC_Bactec from the equine isolate had a size of 92,970 bp and belonged

to the incompatibility group IncI1. In contrast, the plasmids from isolates of human

origin belonged to the replicon type FII and had a size of 73,801 bp (pEC_B24) or

118,525 bp (pEC_L8) while the third, plasmid pEC_L46, had two replicons, namely

FII and FIA, and a size of 144,871 bp (Smet et al., 2010). The same was shown for

IncF plasmids of varying sizes from Australia (Partridge et al., 2011) and in Germany,

a human clinical E. coli ST131 isolate harboured an IncFII plasmid (pKCT407) with

the �orf477-�tnpA structure (Cullik et al., 2010). A 220,824 bp plasmid from Swedish

clinical human K. pneumonia isolates is believed to have obtained a blaCTX-M-15 region

similar to pEK499 and pC15-1a by recombination events (Sandegren et al., 2012).

Furthermore, in Acinetobacter baumannii a transposon TnAb15, which harboured

an ISEcp1-blaCTX-M-15-�orf477-tnpA-�IS26 segment, has been identified in the

chromosomal DNA (Potron et al., 2011). A preferred insertion of ISEcp1 into the tnpA

gene of Tn3-like transposons has been proposed (Smet et al., 2010), but according

to Bailey et al. (2011) the Tn3-like transposon was annotated incorrectly and should

be renamed as Tn2-like. The same arrangement could be found in the nucleotide

sequence from a Russian Serratia liquefaciens strain (GenBank accession no.

HM470254) downstream blaCTX-M-22, emphasising tnpA of transposon Tn2 as

favoured insertion site. On the IncI1 plasmid pKC390 (Cullik et al., 2010), the �orf477

is followed by a �mrx gene, pointing towards other insertion sites or recombination

events in which the putative inverted repeat of ISEcp1 might play a role. However,

the occurrence of the ISEcp1-blaCTX-M-15-�orf477-�tnpA structure on different

plasmids and in various members of the family Enterobacteriaceae of either human

or animal origin from different countries underlines the impact of transposition and

recombination events in the spread of the resistance gene blaCTX-M-15.

Table 1: blaCTX-M-carrying plasmids

Plasmid (s) �-lactamase replicon type

size (bp) species host species

country year of isolation

accession no.

reference

pCTX913 blaCTX-M-15 F ~50,000 E. coli dog Germany 2004 FR828676 Schink et al., 2011

e.g. pJIE098 blaCTX-M-15 F ~145,000 E. coli human Australia 2005-2006 EU418920 Partridge et al., 2011

pC15-1a blaCTX-M-15 FII 92,353 E. coli human Canada 2000 AY458016 Boyd et al., 2004

pEK516 blaCTX-M-15 FII 64,471 E. coli human United Kingdom nd EU935738 Woodford et al., 2009

pEC_B24 blaCTX-M-15 FII 73,801 E. coli human Belgium nd GU371926 Smet et al., 2010

pKCT407 blaCTX-M-15 FII nd E. coli human Germany 2006 GQ274935 Cullik et al., 2010

pEC_L8 blaCTX-M-15 FII, FIA 118,525 E. coli human Belgium nd GU371928 Smet et al., 2010

pEC_L46 blaCTX-M-15 FII, FIA 144,871 E. coli human Belgium nd GU371929 Smet et al., 2010

pEK499 blaCTX-M-15 FII, FIA 117,536 E. coli human United Kingdom nd EU935739 Woodford et al., 2009

pUUH239.2 blaCTX-M-15 FIIK/FII 220,824 K. pneumoniae human Sweden nd CP002474 Sandegren et al., 2012

pEC_Bactec blaCTX-M-15 I1 92,970 E. coli horse Belgium nd GU371927 Smet et al., 2010

pKC390 blaCTX-M-15 I1 nd E. coli human Germany 2006 GQ274928 Cullik et al., 2010

pYC-5b blaCTX-M-15 nd ~50,000 E. coli human Cameroon 2002 AY604721 Gangoue-Peboji et al., 2005

pMRC151 blaCTX-M-15 nd >58,000 K. pneumoniae human Nigeria 2002-2003 AY995205 Soge et al., 2006

several blaCTX-M-15 nd nd E. coli human France 2001 AM040706 Eckert et al., 2006

pCTX168 blaCTX-M-1 N ~50,000 E. coli dog Germany 2004 FN806788 Schink et al., 2011

pCTX246 blaCTX-M-1 N ~50,000 E. coli swine Germany 2004 FN806790 Schink et al., 2011

p1204y1463 blaCTX-M-1 N ~130,000 E. coli human Spain 2004 FJ235692 Diestra et al., 2009

pKC394 blaCTX-M-1 N 53207 E. coli human Germany 2006 HM138652 Cullik et al., 2010

nd blaCTX-M-1 N ~40,000 E .coli human Spain 2001-2002 nd Novais et al., 2007

several blaCTX-M-1 N ~45,000 E. coli human, swine

Denmark 2006-2007 nd Moodley & Guardabassi, 2009

several blaCTX-M-1 N ~30,000 E. coli horse Czech Republic 2008 nd Dolejska et al., 2011a

several blaCTX-M-1 N ~40,000 E. coli cattle Czech Republic 2008 nd Dolejska et al., 2011b

nd blaCTX-M-1 N ~35,000 E. coli mallard Poland 2008-2009 nd Literak et al., 2010

pKCT398 blaCTX-M-1 I1 nd E. coli human Germany 2006 GQ274931 Cullik et al., 2010

several blaCTX-M-1 nd nd E. coli human France 2002 AM003904 Eckert et al., 2006

Dis

cu

ssio

n C

ha

pte

r 4

- 21

-


- 22 -

ISEcp

1

bla C

TX-M

-15

�or

f477

�tn

pATn

2

pCTX913

ISEcp

1

bla C

TX-M

-15

orf4

77

unnamed plasmid

ISEcp

1

bla C

TX-M

-15

�or

f477

�tn

pATn

2

pC15-1apEK516pEC_B24pEC_L8pEC_L46pEC_BactecpYC-5bpKCT407pJIE098

�tn

pATn

2

�IS

Ecp

1bl

a CTX-

M-1

5�or

f477

�tn

pATn

2

IS26

pKC390bl

a CTX

-M-1

5�or

f477

�tn

pATn

2

IS26

pEK499

�IS

Ecp

1bl

a CTX

-M-1

5�or

f477

�tn

pATn

2

IS26

pUUH239.2

FR828676

AM040706

AY458016EU935738GU371926GU371928GU371929GU371927AY604721GQ274935EU418920

CP002474

GQ274928

EU935739

1000 bp1000 bp

On pCTX168 and pCTX246 the insertion sequence ISEcp1 was truncated by an

IS26 element. This IS26-�ISEcp1 structure was also reported on IncN plasmids from

Spain and Germany and seems to be widely distributed (Diestra et al., 2009; Cullik et

al., 2010).

In the downstream region of the blaCTX-M-1 gene on pCTX168 and pCTX246, a

truncated mph(A)-mrx-mphR(A) gene cluster was detected. A complete mph(A)-mrx-

Figure 1: (a) Schematic presentation of the genetic environment of blaCTX-M-15 on pCTX913. (b) Schematic presentation of genetic environments of blaCTX-M-15. The open reading frames are shown as arrows, with the arrowhead indicating the direction of transcription. IS elements are shown as boxes. Homologous regions are indicated by grey shading.

(a)

(b)


- 23 -

mphR(A) gene cluster was described by Noguchi et al. (2000) in E. coli. The

mphR(A) gene and the terminal 55 bp of mrx were absent. This truncation could be

due to either an interplasmid recombination event or the insertion of an intact ISEcp1,

because the putative recombination site resembles also a putative inverted repeat of

ISEcp1. Whether the ISEcp1-blaCTX-M-1-�orf477 transposon inserted into mrx and an

IS26 subsequently truncated the ISEcp1 or whether this structure had been acquired

by interplasmid recombination events cannot be determined in retrospect.

IS26�IS

Ecp

1bl

a CTX

-M-1

�or

f477

�m

rx

�m

ph(A

)or

f

IS26�IS

Ecp

1bl

a CTX

-M-1

�or

f477

�m

rx

mph

(A)

IS26

pCTX246

pCTX168

ISEcp

1

orf4

77

unnamed plasmid

bla C

TX-M

-1

IS26�IS

Ecp

1bl

a CTX-

M-1

�or

f477

�m

rx

mph

(A)

IS26

pKC398

IS26�IS

Ecp

1bl

a CTX

-M-1

�or

f477

�m

rx

mph

(A)

IS26

pKC394

IS26�IS

Ecp

1bl

a CTX

-M-1

p1204y1463

FN806790

FN806788

AM003904

FJ235692

GQ274931

GQ274929

1000 bp1000 bp

Figure 2: (a) Schematic presentation of the genetic environment of blaCTX-M-1 on pCTX168 and pCTX246. (b) Schematic presentation of genetic environments of blaCTX-M-1. The open reading frames are shown as arrows, with the arrowhead indicating the direction of transcription. IS elements are shown as boxes. Homologous regions are indicated by grey shading.

(a)

(b)


- 24 -

Upstream of the �mrx gene, a complete mph(A) gene followed 45 bp afar by a

complete insertion sequence IS26 with the same orientation as the IS26 upstream of

blaCTX-M-1 was detected on pCTX246. The structure IS26-�ISEcp1-blaCTX-M-1-�orf477-

�mrx-mph(A)-IS26 has been described on IncN plasmids (e.g. pKC394) from

German human E. coli ST131 isolates, but the second IS26 was 3 bp upstream of

mph(A), whereas on an IncI1 plasmid, pKC398, from German E. coli ST398 the

second IS26 was also 45 bp upstream of mph(A) (Cullik et al., 2010). The authors

have proposed a novel composite transposon, with two equally oriented IS26 and the

exchange of large blaCTX-M-1-containing modules between different plasmid

backbones (Cullik et al., 2010), but the IS26-�ISEcp1-blaCTX-M-1-�orf477-�mrx-

mph(A)-IS26 structures on plasmids pKC394 and pKC398 probably developed

independently because of the dissimilar spacers between mph(A) and IS26. The

occurrence of the same IS26-�ISEcp1-blaCTX-M-1-�orf477-�mrx-mph(A)-IS26 on IncN

and IncI1 plasmids might support the theory of IS26 mediated transposition events

between different plasmid backbones.

However, on pCTX168 a truncated mph(A) gene was identified upstream of �mrx

and a second IS26 was not detected within the sequenced part of the plasmid. The

truncation might be due to interplasmid recombination events. Despite this difference,

the blaCTX-M-1 gene regions on plasmids pCTX246 and pCTX168 are otherwise

related. Comparisons with sequences in the database of the National Center for

Biotechnology Information (NCBI) revealed that this blaCTX-M-1 region has been

identified only on plasmids of German origin so far. The corresponding E. coli

isolates originated from farm and companion animals as well as from humans,

pointing towards an extensive distribution in Germany. Interestingly, the animal

E. coli isolates had been obtained in 2004, while the human isolates were from 2006

(Cullik et al., 2010), but whether this blaCTX-M-1 gene region has developed in animal

isolates first is speculative.

The gene blaCTX-M-1 has been described in conjunction with IncN plasmids of ca. 40

kb in E. coli isolates of human origin from Spain (Novais et al., 2007). Furthermore, it

was found on ca. 45 kb IncN plasmids in porcine and human E. coli isolates from

Denmark (Moodley & Guardabassi, 2009), on 40 kb IncN plasmids in E. coli from


- 25 -

cattle as well as on 30 kb IncN plasmids of equine origin from the Czech Republic

(Dolejska et al., 2011a; Dolejska et al., 2011b). A 35 kb blaCTX-M-1-carrying IncN

plasmid has been identified in an E. coli isolate from a polish wild mallard (Literak et

al., 2010). Thus, the blaCTX-M-1 gene appears to be widely distributed on IncN

plasmids of different sizes but there is no further information about the immediately

flanking regions, except the linkage with ISEcp1 upstream of blaCTX-M-1 on the

Spanish plasmids (Novais et al., 2007).

Moreover, blaCTX-M-1 has been identified on plasmids of various incompatibility

groups. The aforementioned IncI1 plasmids have been detected aside from Germany

(Cullik et al., 2010) in the Czech Republic (Dolejska et al., 2011a), in Poland (Literak

et al., 2010) and in France and the Netherlands from E. coli isolates of healthy

poultry (Girlich et al., 2007). In addition, IncI1 plasmids carrying blaCTX-M-1 have been

reported from the Netherlands in E. coli isolates from human, poultry and retail meat

(Leverstein-van Hall et al., 2011).

3. Occurrence of the qnrB19 gene in Germany and in other countries

The first detection of a qnrB19 gene has been on plasmid pR4525 from a clinical

human E. coli isolate from Colombia obtained in 2002 (Cattoir et al., 2008a). Later on

the gene was reported in a clinical human K. pneumoniae isolate from the United

States identified in 2007 (Endimiani et al., 2008). In a Salmonella enterica serovar

Typhimurium isolate, obtained in 2004 from a case of human gastroenteritis in Italy, a

qnrB19 carrying plasmid has been identified (Dionisi et al., 2009). Furthermore,

qnrB19 has been detected in human S. Typhimurium isolates from the Netherlands

(García-Fernández et al., 2009; Hammerl et al., 2010; Veldman et al., 2011) and in

reptile Salmonella spp. isolates from Germany (Dierikx et al., 2010, Guerra et al.,

2010) as well as in human Salmonella isolates from Korea (Jeong et al., 2011). In

another study, the gene was found in human commensal E. coli, K. pneumoniae and

Escherichia hermannii from Peru and Bolivia (Pallecchi et al., 2010). In Spain, a

qnrB19-positive human E. coli has been isolated in 2005 (Rios et al., 2010). In


- 26 -

addition, qnrB19 has been identified in the Netherlands in a bovine E. coli isolate

(Hordijk et al., 2011) and in equine E. coli from the Czech Republic (Dolejska et al.,

2011a).

In an international collaborative retrospective study, the occurrence of PMQR

genes in S. enterica and E. coli was investigated. The qnrB19 gene was detected in

S. enterica isolates from humans and fowl from the Netherlands, human isolates from

the UK and turkey isolates from Denmark and Finland. In Germany, qnrB19 has been

identified in S. enterica isolates from food, fowl, turkeys and reptiles. In E. coli

isolates, qnrB19 has been solely detected among those from Polish turkeys

(Veldman et al., 2011). The qnrB19 gene has also been identified in Salmonella

enterica serovar Corvallis isolates from poultry in Brazil (Ferrari et al., 2011) as well

as in E. coli isolates from chicken in Nigeria (Fortini et al., 2011).

The E. coli isolates harbouring pQNR2078 and pQNR2086 originated from mares

suffering from genital tract infections and were identified in 2005 in Germany. This is

the first report of qnrB19 in E. coli of equine origin in Germany. The qnrB19 gene has

been basically detected worldwide in different members of the family

Enterobacteriaceae of human and animal origin, including commensal isolates and

isolates from food and food-producing animals.

Both equine E. coli isolates had the sequence type, ST86. In the MLST-database

are entries of E. coli ST86 of bovine and simian origin from Egypt and the U.S.A.,

repectively (http://mlst.ucc.ie/mlst/dbs/Ecoli, last accessed 15 March 2012).

Furthermore, E. coli ST86 has been identified among ESBL-producing E. coli isolates

from seagulls in Portugal (Simões et al., 2010).

4. The genetic environment of the qnrB19 gene

The qnrB19 gene has been described to be located on plasmids of varying sizes

and replicon types, which are listed in Table 2. Figure 3 shows schematically the

genetic environment of the gene qnrB19. The first qnrB19 gene described was

located on the 40 kb plasmid pR4525 and organised within a 2,739 bp novel


- 27 -

transposon Tn2012, consisting of ISEcp1C and qnrB19 (Cattoir et al., 2008a).

Unfortunately, the incompatibility group of pR4525 has not been mentioned.

Almost at the same time, an 80 kb plasmid (pLRM24) containing a large composite

genetic element, designated KQ element, from a human K. pneumoniae isolate from

the U.S.A. has been reported (Rice et al., 2008). The qnrB19 gene was part of the

novel transposon Tn5387 of 2,966 bp in size, which was located in close proximity to

the blaKPC-3 carrying transposon Tn4401. Tn5387 consists of qnrB19 and an ISEcp1,

which showed a single basepair exchange in comparison to the ISEcp1 of Tn2012.

The region between ISEcp1 and qnrB19 showed 100% identity in both transposons,

but the sequence upstream of qnrB19 differed. In Tn2012 qnrB19 was 157 bp afar

from the end of the putative inverted repeat of ISEcp1, whereas in Tn5387 it was 383

bp. The first 155 bp showed 100% identity, including 14 bp of the putative inverted

repeat of Tn2012.

On the IncL/M-like plasmid p61/9 from a S. enterica isolate from Italy, the

transposon Tn2012 was followed downstream by an IS26 bracketed region

containing blaSHV-12. The authors had proposed that the acquisition of Tn2012 on

p61/9 was due to an illegitimate recombination, because the 5 bp target site

duplication after the second right inverted repeat was absent (Dionisi et al., 2009).

However, the ISEcp1 showed the same exchange in the nucleotide sequence as the

one in Tn5387 and the first 246 bp upstream qnrB19 were also identical, whereas the

similarity with Tn2012 ended 155 bp upstream qnrB19. This raised the question

whether Tn2012 had been inserted into p61/9. A closer look at the sequence

revealed that the last homologous base pairs of p61/9 with Tn5387 resembled a

putative inverted repeat of ISEcp1 with 8 of 14 base pairs identity (Poirel et al.,

2005). The complete homologous region started and ended with inverted repeats and

was flanked by 5 bp direct repeats. Thus, another putative transposon containing

qnrB19 seems to be present on p61/9. (Fig.3b)

Table 2: qnrB19-carrying plasmids

plasmid (s) replicon type size (bp) species host species

country year of isolation

accession no.

reference

pQNR2078 N 42,379 E. coli horse Germany 2004 HE613857 Schink et al., 2012

pR4525 nd ~40,000 E. coli human Colombia 2002 EU523120 Cattoir et al., 2008a

pLRM24 nd ~80,000 K. pneumoniae human U.S.A. 2007 EU624315 Rice et al., 2008

p61/9 L/M nd S. enterica human Italy 2004 FJ790886 Dionisi et al.,2009

pSGI15 ColE-like 2699 S. enterica human Netherlands nd FN428572 Hammerl et al., 2009

pECY6-7 ColE-like 2699 E. coli human Peru 2005 GQ374156 Pallecchi et al., 2010

pECC14-9 ColE-like 3071 E. coli human Bolivia 2005 GQ374157 Pallecchi et al., 2010

several ColETp (n=7) 2700-4500 Salmonella spp. reptile Germany 2000-2008 nd Guerra et al., 2010

pSR132 nd >23,000 E. coli human Italy 2007-2008 GU074393 Richter et al.,2010

p013.1IncR R ~40,000 E. coli cattle Netherlands nd HM146784 Hordijk et al., 2011

several N (n=2) ~40,000 E. coli horse Czech Republic

2008 nd Dolejska et al., 2011a

Ch

ap

ter 4

Dis

cu

ssio

n

- 28

-


- 29 -

�tn

pA

Tn172

1

IS26

qnrB

19

IS26 �

orf1

Tn172

1

pQNR2078/2086

pR4525

Tn2012Tn2012

qnrB

19

ISEcp

1C

IRL: CCTAGATTCTACDR: ATCAA

IRR1: ACGTGGAATTTAGG

IRR2: ACCCAGTAATTCAGDR: ATCAA

pLRM24

Tn5387

qnrB

19

ISEcp

1

�or

f1

Tn172

1

�or

f1

Tn172

1

IRL: CCTAGATTCTACDR: GAATT

IRR2: ACGTAGAAAATAGGDR: GAATT


�bla O

XA-9

�bla O

XA-9

p61/9

qnrB

19

ISEcp

1

IRL: CCTAGATTCTACDR: TTTTT


IS26

pSGI15pECY6-7

qnrB

19

p013.1IncR

qnrB

19

IS26

IS26

pSR132

qnrB

19

HE613857

EU523120

EU624315

FJ790886

FN428572GQ374156

GU074393

HM146784

1000 bp1000 bp

IRR2: ACGCAGATCCAGCGDR: TTTTT

putative transposon

Figure 3: (a) Schematic presentation of the genetic environment of qnrB19 on pQNR2078 and pQNR2086. (b) Schematic presentation of genetic environments of qnrB19. The open reading frames are shown as arrows, with the arrowhead indicating the direction of transcription. IS elements are shown as boxes. Homologous regions are indicated by grey shading.

(a)

(b)


- 30 -

The 2699 bp ColE-like plasmid pSGI15, harboured by a human S. enterica isolate

from the Netherlands, has been described in 2009 (Hammerl et al., 2010). The

immediate flanking regions of qnrB19, 245 bp upstream and 225 bp downstream,

showed 100% identity with Tn5387 and the qnrB19 region on p61/9. This plasmid

exhibited 100% identity with pECY6-7, which had been identified in a study on

human commensal E. coli isolates from Peru and Bolivia, as well as in K.

pneumoniae and E. hermannii (Pallecchi et al., 2010). In the same study, the

structurally related ColE-like plasmid pECC14-9 of 3071 bp, which carried an

identical qnrB19 region, was described. Hence ColE-like plasmids carrying qnrB19

seem to have widely spread geographically and within the Enterobacteriaceae. This

is underlined by the finding of small ColETp plasmids, carrying qnrB19 from

Salmonella spp. of reptile origin isolated in Germany (Guerra et al., 2010)

The qnrB19 gene has been also identified within an ISCR1 complex class 1

integron in an E. coli isolate from Italy (Richter et al., 2010). The sequence deposited

in the database showed 100% identity with Tn5387 in the first 221 bp immediately

up- and 225 bp downstream of qnrB19.

From the Netherlands, the 40 kb plasmid p013.1IncR, harboured by a bovine E.

coli isolate, has been reported. This plasmid showed a similar upstream region of

qnrB19 in comparison to the aforementioned plasmid p61/9. However, instead of an

insertion sequence ISEcp1 another IS26 was located 44 bp downstream qnrB19 on

this IncR plasmid (Hordijk et al., 2011). The upstream region of qnrB19 on

pQNR2078 and pQNR2086 showed also similarity with p61/9 and 140 bp

downstream of qnrB19, a second IS26 in the same orientation was identified. These

140 bp had 100% identity with Tn2012 and Tn5387. The insertion of IS26 results in 8

bp direct repeats in the target side either flanking a single IS26 or if two direct

repeated IS26 act as a transposon flanking both (Iida et al., 1984). As the 8 bp

immediately up- and downstream of the two IS26 were entirely different, the

formation of this region was probably not due to integration of IS26 but to

recombination events involving these insertion sequences. However such a region

might act as a transposon in the future. The dissimilar spacers between qnrB19 and


- 31 -

the downstream IS26 on pQNR2078/2086 and p013.1IncR point towards an

independent development of these genetic structures.

In the Czech Republic, 40 kb conjugative IncN plasmids carrying solely qnrB19

have been isolated from E. coli of equine origin (Dolejska et al., 2011a). These

plasmids resembled in size the 42,379 bp plasmid pQNR2078 from equine E. coli

sequenced in the present PhD study. Therefore, such plasmids seem to be widely

distributed among E. coli isolates from horses.

Remarkably, the qnrB19 gene was the only resistance gene on plasmids

pQNR2078/2086. In other studies qnrB19 has been co-located with other resistance

determinants like ESBL genes. On the 40 kb plasmid pR4525, blaCTX-M-12 and blaSHV-

12 have been located in addition to qnrB19 and in another study a blaSHV-12 gene has

been detected on a qnrB19-carrying plasmid (Cattoir et al., 2008a; Rios et al., 2010).

These findings indicate that the genetic environment of the qnrB19 genes is involved

in ongoing alterations due to transposition and recombination events and similar

structures might be found in different members of the family Enterobacteriaceae.

5. Plasmids in Enterobacteriaceae

The spread of resistance genes is on one hand due to successful clones, which

could emerge all over the world, and on the other hand mediated by horizontal gene

transfer in which plasmids play an important role. Plasmids are classified in

incompatibility groups and 27 Inc groups have been described in Enterobacteriaceae

so far (Carattoli, 2011).

Plasmids of the IncF family are widespread among Enterobacteriaceae but also

restricted to them (Carattoli, 2011). They play a major role in the dissemination of

blaCTX-M-15 genes and are frequently found in association with the successful E. coli

clone O25:H4-ST131, but also in other E. coli lineages (Carattoli, 2009; Lopéz-

Cerero et al., 2011; Partridge et al., 2011; Smet et al., 2010; Woodford et al., 2009).

In addition, the gene aac(6’)-Ib-cr has been described to be co-located on these


- 32 -

plasmids. Plasmid pCTX913 carried an aac(6’)-Ib-cr gene as well (unpublished data),

indicating that it was closely related to these widespread and successful plasmids.

Among the plasmid families, the IncN type is widely distributed among

Enterobacteriaceae worldwide (Carattoli, 2009) and exhibits a broad host range

(Krishnan & Iyer, 1988). IncN plasmids have been reported carrying different

resistance genes e.g. blaCTX-M-1, blaCTX-M-3, blaCTX-M-15 and blaCTX-M-65 (Cullik et al.,

2010; Dolejska et al., 2011a; Dolejska et al., 2011b; Literak et al., 2010; Marcadé et

al., 2009; Moodley & Guardabassi, 2009; Novais et al., 2007) and also different qnr

genes like qnrB2, qnrB19 and qnrS1 (García-Fernández et al., 2009). A plasmid

multilocus sequence typing scheme has been established to categorise IncN

plasmids in different sequence types based on the nucleotide sequence of selected

loci within the genes repA, traJ and korA in order to discriminate IncN plasmids more

precisely (García-Fernández et al., 2011). IncN plasmids carrying blaCTX-M-1 and

exhibiting pMLST type ST1 have been identified in E. coli isolates from human and

animal isolates from Greece, Italy, Denmark, the Netherlands, Germany and the UK

(http://pubmlst.org/ plasmid/, last accessed 15 March 2012).

The plasmids pCTX168 and pCTX246 also belonged to ST1 and have been

deposited as ESBL-248 (id 568) and ESBL-249 (id 569), respectively, in the

PubMLST database. In contrast, one of the aforementioned blaCTX-M-1-carrying IncN

plasmids, pKC394, from German human E. coli isolates with a similar genetic

arrangement immediately up- and downstream of blaCTX-M-1 (Cullik et al., 2010) as

pCTX246 has been completely sequenced and revealed the pMLST type ST8. The

same pMLST type was identified for the blaCTX-M-65 carrying IncN plasmid pKC396

(García-Fernández et al., 2011). ST1 differs from ST8 in three nucleotides within

repN and in one nucleotide within traJ. The finding of similar genetic environments of

blaCTX-M-1 on different IncN plasmids points towards gene transfer either by

recombination events or by transposition between different IncN plasmid backbones

as already proposed (Cullik et al., 2010). Another possibility would be the alteration

of the pMLST specific loci on the plasmid itself.

The pMLST type ST8 had been determined for plasmids pQNR2078/2086 as well

and the nucleotide sequence of pQNR2078 had a remarkable overall identity of 99.0


- 33 -

% with plasmid pKC396. Both sequences differed only in the resistance gene region

and within their iterons. It is possible that the IncN pMLST ST8 backbone acquired

different resistance gene regions by independent recombination events potentially

involving IS26.

Furthermore, a human S. Typhimurium isolate from the Netherlands harboured an

IncN-ST8 plasmid that carried qnrB19 (García-Fernández et al., 2009). This

observation indicated a wide distribution of those plasmids geographically and within

the family Enterobacteriaceae. However, no further information about the size of the

plasmid and the nucleotide sequence is available and therefore it would be too

speculative to conclude that it could be the same plasmid as pQNR2078/2086.

The localisation of ESBL and qnr genes on related IncN plasmids and linked to

insertion sequences like ISEcp1 and especially IS26 is a cause of concern as IncN

plasmids have been described to be easily transmitted between different E. coli

lineages of human and animal origin and among members of different species within

the family Enterobacteriaceae (Krishnan & Iyer, 1988; Moodley & Guardabassi,

2009). Moreover, similar genetic structures and IS elements facilitate homologous

recombination events, resulting in a genetic environment of the blaCTX-M-1 and the

qnrB19 genes that represents an appropriate basis for further dissemination.

6. Animal reservoirs?

It has been stated that the transmission of ESBLs is more likely due to plasmid-

mediated horizontal gene transmission than due to the expansion of bacterial clones

(Carattoli, 2008). The transmission of IncN plasmids carrying blaCTX-M-1 from E. coli

isolates of porcine origin to different E. coli lineages found among farmworkers

(Moodley, 2009) and the in situ conjugation of blaTEM-52 carrying IncI1 plasmids from

poultry to human commensal E. coli (Smet et al., 2011) support this theory. As qnr

genes have been detected on the same plasmid types, they have theoretically similar

transmission potentials and the finding of an IncN-ST8 plasmid, which carried


- 34 -

qnrB19, in a human S. Typhimurium isolate (García-Fernández et al., 2009) might

point towards plasmid transmission.

There are several reports of blaCTX-M and qnr genes in commensal and pathogenic

bacteria of livestock (Aarestrup et al., 2006; Agerso et al., 2011; Blanc et al., 2006;

Briñas et al., 2005; Dolejska et al., 2011b; Fortini et al., 2011; Girlich et al., 2007;

Gonçalves et al., 2010; Kirchner et al., 2011; Liu et al., 2008; Meunier et al., 2006;

Richter et al., 2010; Rodríguez-Martínez et al., 2001; Smet et al., 2008; Veldman et

al., 2011; Yao et al., 2011) and companion animals (Carattoli et al., 2005; Costa et

al., 2004; Costa et al., 2008; Dolejska et al., 2011b; Maddox et al., 2011a; Shaheen

et al., 2011; Wittum et al., 2010). These findings underline that ESBL-producing and

qnr-positive isolates are involved in animal diseases. However, the reports of these

genes in isolates from healthy animals are of concern, because they could enable

unnoticed transmission of resistance determinants. Transmission between animals in

the same barn has been described for horses from the UK, and thus document the

spread of bacteria with resistance genes in the animal population (Maddox, 2011b).

In addition, such genes have been detected in bacterial isolates from wild animals,

indicating another potential reservoir of these resistance genes in animals (Guenther

et al., 2011; Literak et al., 2010).

Community-acquired infections with ESBL-producing E. coli have been reported

and the authors proposed that retail meat could be a source of such isolates, which

then could cause infections, colonise the human gut or donate resistance plasmids

(Doi et al., 2010). This theory is supported by a study from the Netherlands in which

blaCTX-M-1 genes have been identified on IncI1-ST7 plasmids within E. coli ST58 and

ST117 isolates from poultry, retail meat and human patients suggesting transmission

of ESBL-producing E. coli through the food chain (Leverstein-van Hall et al., 2011).

Companion animals live nowadays in close contact with their owner and receive

intensive care, thus facilitating the transmission of bacteria from humans to animals

and vice versa (Guardabassi et al., 2004; Wieler et al., 2011). The occurrence of

CTX-M-15 ESBL-producing E. coli of the O25:H4-ST131 group among dogs from the

Netherlands, France, Denmark, Spain and Germany, as well as in a horse (Ewers et

al., 2010) points towards clonal transmission from human to animals, which could


- 35 -

also be possible for other bacteria, such as E. coli ST410, besides plasmidic spread

of resistance genes. The occurrence of ESBL and qnr genes in wild animals might be

due to ingestion of contaminated human waste via e.g. wastewater (Martinez, 2009)

or land-application of livestock manure (Guenther et al., 2011; Kümmerer, 2009).

Yellow-legged gulls feeding at a city dump in France harboured ESBL-producing

Enterobacteriaceae (Bonnedahl et al., 2009). Recently, human waste-associated

CTX-M-1 and CTX-M-15 ESBL-producing E. coli from Antarctic water samples have

been identified underlining the human impact on environmental contamination with

resistant bacteria (Hernández et al., 2012).

Several authors stated the presence of an animal reservoir after detecting ESBL

genes in animal isolates (Bonnedahl et al., 2009; Geser et al., 2011; Girlich et al.,

2007; Leverstein-van Hall et al., 2011; Smet et al., 2008). Moreover, a reservoir in

healthy humans has been considered (Ben Sallem et al., 2011). Nevertheless, the

occurrence of related plasmids and distinct clones in humans as well as in animals

points towards transmission between different host species, although there is no

proof for a stable reservoir among animals so far.

7. Concluding remarks

The number of ESBL-producers as well as qnr-positive isolates among the 417 E.

coli isolates of the BfT-GermVet study was low.

The ESBL genes blaCTX-M-1 and blaCTX-M-15 and the PMQR gene qnrB19 were

detected in E. coli isolates from diseased animals in Germany. Several reports

described the co-localisation of ESBL and PMQR genes within the same isolate and

on the same plasmid (Dionisi et al., 2009; Dolejska et al., 2011a; Kirchner et al.,

2011; Müller et al., 2011; Richter et al., 2010; Woodford et al., 2009; Yao et al.,

2011). In contrast, the porcine and canine isolates, harbouring pCTX168 and

pCTX246 with blaCTX-M-1, were not positive for qnr or aac(6’)-Ib-cr genes and the

equine isolates, harbouring pQNR2078 and pQNR2086 with qnrB19, did not produce

an ESBL. Solely one of the five investigated plasmids pCTX913 from a canine E. coli


- 36 -

isolate carried an aac(6’)-Ib-cr and a blaCTX-M-15 gene. These findings and the in depth

analysis of the plasmids harbouring these genes contribute to the knowledge of the

distribution and organisation of such resistance genes.

- 37 -

Chapter 5

Summary

Chapter 5 Summary

- 38 -

Summary

Anne-Kathrin Schink Studies on the prevalence, distribution and organization of

extended-spectrum �-lactamase genes and transferable

(fluoro)quinolone resistance genes among Enterobacte-

riaceae from defined disease conditions of companion and

farm animals

Extended-spectrum �-lactamase (ESBL)- and Qnr-protein-producing Escherichia coli

isolates have gained considerable public attention during recent years. However,

information about such isolates from diseased animals in Germany is scarce. Thus,

the aims of this study were (i) to determine how often and which subtypes of ESBL

and qnr genes are present in E. coli from defined disease conditions of companion

and farm animals and (ii) to gain insight into the location and organization of the

resistance genes. For this, the E. coli isolates collected all over Germany in the BfT-

GermVet study were used.

In the BfT-GermVet study, 417 E. coli isolates from diseased dogs/cats (n = 228),

horses (n = 102), and swine (n = 87) were tested for their susceptibility to 24

antimicrobial agents by broth microdilution. To identify potential ESBL-producers, all

100 ampicillin-resistant E. coli isolates from this collection were subjected to an initial

screening for cefotaxime resistance and subsequent phenotypic confirmatory tests.

In a second part of the project, all E. coli isolates were screened for qnr genes. The

ESBL and qnr genes were detected by specific PCR assays and the complete

resistance genes including their flanking regions were cloned and sequenced.

Plasmids were transferred by conjugation and transformation into E. coli recipients

and subjected to PCR-based replicon typing. One qnrB19-carrying plasmid was

sequenced completely. Multilocus sequence typing (MLST) was performed for the

ESBL-producing and for the qnr-positive E. coli isolates.

Summary Chapter 5

- 39 -

Solely three E. coli isolates showed an ESBL phenotype. The canine isolate 913

from a urinary tract infection harboured a blaCTX-M-15 gene and the E. coli isolates

from canine pneumonia (isolate 168) and porcine metritis-mastitis-agalactia

syndrome (isolate 246) blaCTX-M-1 genes. The gene qnrB19 was detected in two E.

coli isolates (2078 and 2086) from mares suffering from genital tract infections.

MLST showed that isolate 913 had the sequence type ST410, while isolates 168

and 246 belonged to the novel sequence types ST1576 and ST1153, respectively.

Isolates 2078 and 2086 were assigned to ST86.

Isolate 913 harboured the ca. 50 kb IncF plasmid pCTX913. This plasmid carried

the blaCTX-M-15 gene and an aac(6’)-Ib-cr gene, which confers resistance to kanamycin

and reduced susceptibility to ciprofloxacin. Furthermore, pCTX913 conferred

resistance to gentamicin and tetracycline. Upstream of blaCTX-M-15 the insertion

sequence ISEcp1 was present and downstream a truncated orf477 and a fragment of

a transposase gene tnpA.

The two blaCTX-M-1 ESBL genes were located on structurally related plasmids of ca.

50 kb which did not confer any other resistance properties. PCR-based replicon

typing identified both plasmids, designated pCTX168 and pCTX246, to belong to

IncN. Plasmid pCTX168 was conjugative. The blaCTX-M-1 upstream and the immediate

downstream regions of pCTX168 and pCTX246 were similar whereas the sequences

further downstream of blaCTX-M-1 differed. In the upstream region, a fragment of the

insertion sequence ISEcp1, truncated by an IS26 element, was detected. In the

downstream region, a fragment of orf477 and a truncated mrx gene were identified

on both plasmids. On plasmid pCTX246, a complete mph(A) gene and another IS26

element were seen further downstream of the �mrx gene, while on plasmid pCTX168

the mph(A) gene was truncated.

The qnrB19-carrying conjugative plasmids pQNR2078 and pQNR2086 belonged

also to IncN, were indistinguishable by restriction analysis and did not carry other

resistance genes. The qnrB19 gene was flanked by copies of the insertion sequence

IS26 located in the same orientation. The completely sequenced plasmid,

pQNR2078, had a size of 42,379 bp and exhibited 47 open reading frames. Except

for the resistance gene region, the remaining part of pQNR2078 showed 99%

Chapter 5 Summary

- 40 -

nucleotide sequence identity to the blaCTX-M-65-carrying plasmid pKC396 from human

E. coli.

The blaCTX-M-15 gene region as identified on the IncF plasmid pCTX193 has been

detected in diverse plasmid backgrounds in different members of the

Enterobacteriaceae from human and animal sources isolated in several countries. In

addition, the gene blaCTX-M-1 gene in combination with the IS26-�ISEcp1-structure,

the truncated mph(A)-mrx-mphR(A) gene cluster which is followed by a second IS26,

linked to IncN plasmids has been described in a German human clinical E. coli

ST131 isolate, but not in isolates of animal origin in Germany so far.

The gene qnrB19 gene was detected in a new genetic environment on conjugative

IncN plasmids and for the first time in an E. coli isolate of equine origin in Germany.

Despite the fact, that the number of blaCTX-M and qnr genes among E. coli from the

BfT-GermVet study was low, the results of this study showed that the integration of

insertion sequences and interplasmid recombination events accounted for the

structural variability of the blaCTX-M gene regions and the formation of the genetic

environment of qnrB19 on pQNR2078. Furthermore, IncN plasmids, which carry

blaCTX-M-1 or qnrB19 genes, might be involved in the dissemination of these

resistance genes among Enterobacteriaceae of animal and human origin.

- 41 -

Chapter 6

Zusammenfassung


- 42 -

Zusammenfassung

Anne-Kathrin Schink Untersuchungen zu Vorkommen, Verbreitung und

Organisation von Genen für �-Laktamasen mit erweiter-

tem Wirkungsspektrum und transferablen (Fluor)Chinolon-

Resistenzgenen bei Enterobacteriaceae aus definierten

Krankheitsprozessen von Kleintieren und Nutztieren

In der jüngeren Vergangenheit sind Escherichia coli-Isolate, die Gene für �-

Laktamasen mit erweitertem Wirkungsspektrum (extended-spectrum �-lactamase;

ESBL) und/oder Qnr-Proteine tragen und so Resistenz gegenüber Penicillinen,

Cephalosporinen und Monobaktamen beziehungsweise (Fluor)Chinolonen zeigen,

zunehmend in den Fokus des öffentlichen Interesses gelangt. Da es über diese

Gene bei E. coli-Isolaten von erkrankten Tieren in Deutschland nur wenig

Informationen gibt, waren die Ziele dieser Studie festzustellen (i) wie häufig und

welche Gene für ESBLs und Qnr-Proteine bei E. coli Isolaten von erkrankten

Kleintieren und Nutztieren zu finden sind und (ii) die genetische Lokalisation und

Organisation dieser Gene zu untersuchen. Hierzu wurde das Stammkollektiv der

deutschlandweit durchgeführten BfT-GermVet Studie verwendet.

In der BfT-GermVet Studie wurden für 417 E. coli-Isolate von erkrankten

Hunden/Katzen (n = 228), Pferden (n = 102) und Schweinen (n = 87) minimale

Hemmkonzentrationen gegenüber 24 antimikrobiellen Wirkstoffen mittels

Bouillonmikrodilution bestimmt. Von diesen 417 E. coli-Isolaten zeigten 100

Resistenz gegenüber Ampicillin. Um potentielle ESBL-Produzenten zu identifizieren,

wurden zusätzlich die minimalen Hemmkonzentrationen für Cefotaxim bestimmt und

anschließend phänotypische Bestätigungstests durchgeführt. Alle E. coli-Isolate

wurden auf das Vorhandensein von qnr-Genen untersucht. Die ESBL- und qnr-Gene

wurden mittels spezifischer PCR-Assays detektiert und die vollständigen Gene

inklusive ihrer flankierenden Regionen kloniert und sequenziert. Plasmide wurden

Zusammenfassung Chapter 6

- 43 -

über Konjugation oder Transformation in E. coli-Empfängerstämme transferiert und

mit PCR-basierter Replikontypisierung charakterisiert. Für die ESBL-produzierenden

und qnr-positiven E. coli-Isolate wurde Multilocus-Sequenztypisierung (MLST)

durchgeführt.

Lediglich bei drei E. coli-Isolaten wurde ein ESBL-Phänotyp festgestellt. Ein Isolat

stammte von einem Hund mit Harnwegsinfektion (Isolat 913), eines von einem Hund

mit Pneumonie (Isolat 168) und das dritte von einer Sau mit Mastitis-Metritis-

Agalaktie Syndrom (Isolat 246). Isolat 913 beherbergte das ESBL-Gen blaCTX-M-15

und Isolate 168 und 246 je ein blaCTX-M-1. In zwei E. coli-Isolaten (Isolate 2078 und

2086) von Stuten mit Genitalinfektionen wurde das Gen qnrB19 identifiziert.

Über MLST wurde für Isolat 913 der bekannte ST410 bestimmt und die Isolate

168 und 246 den neuen Sequenztypen ST1576 und ST1153 zugeordnet. Die Isolate

2078 und 2086 wiesen den Sequenztyp ST86 auf.

Isolat 913 beinhaltete das ca. 50 kb IncF-Plasmid pCTX913 mit den Genen blaCTX-

M-15 und aac(6’)-Ib-cr – letzteres Gen vermittelt Resistenz gegenüber Kanamycin und

verminderte Empfindlichkeit gegenüber Ciprofloxacin. Des Weiteren vermittelte

pCTX913 auch Resistenz gegenüber Gentamicin und Tetrazyklin. In der

stromaufwärts von blaCTX-M-15 gelegenen Region befand sich die Insertionssequenz

ISEcp1 und stromabwärts ein Fragment von orf477 sowie ein Fragment eines

Transposasegenes tnpA.

Die blaCTX-M-1 ESBL-Gene waren auf strukturell ähnlichen ca. 50 kb-großen

Plasmiden der Inkompatibilitätsgruppe IncN lokalisiert (pCTX168 und pCTX246), die

neben der �-Laktamresistenz keine weiteren Resistenzen aufwiesen. Das Plasmid

pCTX168 erwies sich als konjugativ. Der Bereich stromaufwärts von blaCTX-M-1 war

bei beiden Plasmiden gleich und bestand aus einem Fragment der

Insertionssequenz ISEcp1, welche von einer Insertionssequenz des Typs IS26

unterbrochen wurde. Stromabwärts des blaCTX-M-1-Genes wurden ein Fragment des

offenen Leserahmens orf477 und ein partiell deletiertes mrx-Gen ebenfalls auf

beiden Plasmiden gefunden. Weiter stromabwärts unterschieden sich die Sequenzen

der beiden Plasmide dagegen voneinander. Auf pCTX246 wurde ein vollständiges


- 44 -

mph(A)-Gen und ein weiteres IS26 identifiziert, während auf pCTX168 das Gen

mph(A) partiell deletiert war.

Die qnrB19-tragenden IncN-Plasmide pQNR2078 und pQNR2086, erwiesen sich

als konjugativ, waren durch Restriktionsanalysen nicht zu unterscheiden und

vermittelten keine weiteren Resistenzen. Die qnrB19-Gene waren von zwei, in

gleicher Orientierung angeordneten Kopien der Insertionssequenz IS26 flankiert. Das

Plasmid pQNR2078 wurde vollständig sequenziert. Es hatte eine Größe von 42,379

bp und verfügte über 47 offene Leserahmen. Vergleichende Analysen ergaben –

abgesehen von der Resistenzgenregion – eine Identität von 99% zu dem blaCTX-M-65-

tragenden Plasmid pKC396 von einem E. coli-Isolat menschlicher Herkunft.

Die Umgebung des auf dem IncF Plasmid pCTX913 lokalisierten blaCTX-M-15-Genes

ist bereits auf verschiedenen Plasmiden in unterschiedlichen Vertretern der Familie

Enterobacteriaceae menschlichen oder tierischen Ursprungs aus verschiedenen

Ländern beschrieben worden. In E. coli-Isolaten tierischer Herkunft wurde das blaCTX-

M-1 Gen in Kombination mit der IS26-�ISEcp1-Struktur und einem partiell deletierten

mph(A)-mrx-mphR(A)-Genkomplex, dem ein zweites IS26 folgt, auf einem IncN-

Plasmid bisher noch nicht identifiziert. Allerdings wurde diese Struktur bislang bei

einem klinischen E. coli ST131-Isolat von einem Menschen aus Deutschland

nachgewiesen. Das qnrB19-Gen liegt in einem bisher unbekannten genetischen

Kontext auf konjugativen IncN-Plasmiden und wurde im Rahmen dieser Ph.D.-Arbeit

zum ersten Mal bei E. coli-Isolaten von Pferden aus Deutschland identifiziert.

Die Ergebnisse dieser Studie zeigen, dass ESBL- und qnr-Gene bei E. coli-

Isolaten von erkrankten Tieren aus der BfT-GermVet-Studie relativ selten

vorkommen. Weiterhin zeigten die Untersuchungen zur Feinstruktur der

Resistenzgenregionen, dass die Integration von Insertionssequenzen und

Rekombinationen zwischen Plasmiden die strukturelle Variabilität der blaCTX-M-

Regionen und die Entstehung der genetischen Umgebung von qnrB19 auf dem

Plasmid pQNR2078 bedingt haben. Darüber hinaus sind IncN-Plasmide, die Gene

wie blaCTX-M-1 oder qnrB19 tragen, wahrscheinlich an der Verbreitung dieser

Resistenzgene unter Enterobacteriaceae von Menschen und Tieren beteiligt.

- 45 -

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Danksagung

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Danksagung Mein besonderer Dank gilt Herrn Prof. Dr. Stefan Schwarz für die hervorragende und engagierte Betreuung während des Projektes und dass er mir die Niagarafälle gezeigt hat. Frau Kristina Kadlec, Ph.D., danke ich für die Unterstützung und Diskussionen, die mir immer weitergeholfen haben, und dafür, dass sie ihr Büro mit mir und meinem Chaos geteilt hat. Der H. Wilhelm Schaumann Stiftung danke ich für das Stipendium, welches diese Arbeit möglich gemacht hat. Bei Herrn Prof. Dr. Heiner Niemann möchte ich mich für die Bereitstellung des Arbeitsplatzes bedanken. Herrn Prof. Dr. Günter Klein und Herrn Prof. Dr. Peter Heisig danke ich für die Betreuung des Projektes, der Ph.D.-Kommission für die Zulassung und Frau Faber für die gute organisatorische Betreuung. Ich danke allen Kollegen der Arbeitsgruppe von Prof. Schwarz mit denen ich das Vergnügen hatte zu arbeiten für die geduldige Einarbeitung in die verschiedenen Methoden, sowie für die Hilfe und Unterstützung im Labor. Andrea Feßler danke ich für die gemeinsame Ph.D.-Studentenzeit und die harmonischen Kongressreisen. Meinen Eltern, Geschwistern und Freunden danke ich dafür, dass sie immer an mich glauben, für die Seelsorge in Wort und Tat und meinem Vater für die Fütterung der Chinchillas und der Katze, damit ich in der Welt unterwegs sein konnte. Zuletzt möchte ich Tante Heike danken, die mich auf den Weg gebracht hat, ihn aber nicht mit zu Ende gehen konnte.

friedrich-loeffler-institut (fli) institute of farm animal

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