friedrich-loeffler-institut (fli) institute of farm animal
TRANSCRIPT
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.
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, 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 .
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.
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
Chapter 4 Discussion
- 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
-
Chapter 4 Discussion
- 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)
Discussion Chapter 4
- 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)
Chapter 4 Discussion
- 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
Discussion Chapter 4
- 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
Chapter 4 Discussion
- 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
Discussion Chapter 4
- 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
-
Discussion Chapter 4
- 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
IRR1: ACGTGGAATTTAGG
�bla O
XA-9
�bla O
XA-9
p61/9
qnrB
19
ISEcp
1
IRL: CCTAGATTCTACDR: TTTTT
IRR1: ACGTGGAATTTAGG
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)
Chapter 4 Discussion
- 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
Discussion Chapter 4
- 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
Chapter 4 Discussion
- 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
Discussion Chapter 4
- 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
Chapter 4 Discussion
- 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
Discussion Chapter 4
- 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
Chapter 4 Discussion
- 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
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
Chapter 6 Zusammenfassung
- 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.