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Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK I. Rodríguez a,∗ , K. Thomas a , A. Van Essen b , A.-K. Schink c , M. Day d , M. Chattaway d , G. Wu e , D. Mevius b,f , R. Helmuth a , B. Guerra a , on behalf of the SAFEFOODERA-ESBL consortium1 a
Federal Institute for Risk Assessment (BfR), Department for Biological Safety, Max-Dohrn Strasse 8–10, D-10589 Berlin, Germany Central Veterinary Institute (CVI) of Wageningen UR, Department of Bacteriology and TSEs, P.O. Box 65, 8200 AB Lelystad, The Netherlands c Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), 31535 Neustadt-Mariensee, Germany d Public Health England (PHE), 61 Colindale Avenue, London NW9 5EQ, UK e Animal Health and Veterinary Laboratories Agency (AHVLA), Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK f Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands b
a r t i c l e
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Article history: Received 21 October 2013 Accepted 26 February 2014 Keywords: Antimicrobial resistance CTX-M PFGE Plasmids
a b s t r a c t This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n = 356) obtained in Germany, The Netherlands and the UK (2005–2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. -Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 -lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Production of -lactamases is the main mechanism responsible for resistance to -lactams in Enterobacteriaceae. Extendedspectrum -lactamases (ESBLs) are able to hydrolyse third-
∗ Corresponding author. Present address: Department of Microbiology, Hospital Universitario Ramón y Cajal, Carretera de Colmenar Viejo Km 9.100, 28034 Madrid, Spain. Tel.: +34 913 368 152; fax: +34 913 368 809. E-mail address: [email protected] (I. Rodríguez). 1 This consortium also includes: Martin J. Woodward and Nick Coldham, Animal Health and Veterinary Laboratories Agency (AHVLA), UK; Kristina Kadlec and Stefan Schwarz, Friedrich-Loeffler-Institut (FLI), Germany; John Threlfall, Neil Woodford and John Wain, Public Health England (PHE), UK; and Cindy Dierikx, Central Veterinary Institute (CVI), The Netherlands.
and fourth-generation cephalosporins and monobactams, limiting therapeutic options in serious infections caused by Enterobacteriaceae. Over the last decade, the number of ESBL-producing bacteria has increased in many different genera of Enterobacteriaceae and represents a public health threat [1]. CTX-M-type ESBLs are a complex and heterogeneous family of enzymes and may be subdivided into five major groups (CTX-M1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 groups) [2]. These enzymes have spread globally and are the most common ESBLs detected in Enterobacteriaceae, not only in hospitals but also in the community, changing the epidemiology of ESBLs. Among the different CTX-M enzymes, CTX-M-15 (belonging to CTX-M group 1) and CTX-M-14 (belonging to CTX-M group 9) are of high relevance because of their ubiquity, being detected not only in humans and animals but also in environmental samples in many different
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Please cite this article in press as: Rodríguez I, et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.02.019
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countries [1,2]. The successful spread, diversification and maintenance of CTX-M enzymes is due to a combination of different factors: their association with transposable elements and integrons; the capture of these structures by conjugative plasmids; and their transfer to and maintenance in successful bacterial clones [2]. The latter are typified by Escherichia coli clone O25b-ST131, which is a pandemic, multiresistant and uropathogenic lineage frequently associated with the expression of CTX-M-15 and that has contributed significantly to the worldwide spread of this ESBL [3]. This clone habitually harbours the blaCTX-M-15 gene located on plasmids belonging to the IncF family, typically IncFII or multireplicon FII, FIA and FIB [3]. Despite being predominantly plasmid-mediated enzymes, chromosomally located blaCTX-M genes have also been described [4–6]. In this case, the chromosomal location does not enhance the spread of the gene but does assist its stabilisation and maintenance in the bacterium. The objective of this study was to seek and characterise clinical E. coli isolates suspected of expressing chromosomally encoded CTX-M enzymes from Germany, The Netherlands and the UK. 2. Methods 2.1. Bacterial isolates and detection of ˇ-lactamase-encoding genes Within the European SAFEFOODERA-ESBL project (EU ERANet, Ref. 08176), a total of 629 E. coli isolates were selected from the strain collections of the Animal Health and Veterinary Laboratories Agency (AHVLA, UK), Public Health England (PHE, UK), Central Veterinary Institute (CVI, The Netherlands), Friedrich-Loeffler-Institut (FLI, Germany) and Federal Institute for Risk Assessment (BfR, Germany). All isolates were cefotaximenon-susceptible [minimum inhibitory concentrations above the epidemiological cut-off of ≤0.25 mg/L for E. coli, set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); http://www.eucast.org]. Isolates originated from animals (n = 295), animal-derived foods (n = 59), humans (n = 274) and an unknown source (n = 1) and were isolated between 2005 and 2009. These isolates were screened for -lactamase and other resistance genes as well as for virulence determinants using ‘Amr05’ microarrays (ALERE Technologies Ltd., Stirling, UK) in a previous study [7]. For the present work, a subset of 356 isolates was selected
based on the relative contribution of the -lactamase families detected, the source of the isolates and the countries involved. Lactamase genes detected in these isolates with the array were further analysed by PCR sequencing as previously described [8,9]. Plasmid DNA was extracted from the 356 isolates using a Midiprep Plasmid Purification Kit (QIAGEN, Hilden, Germany) and/or the Kado and Liu method [8]. Plasmids were electrotransformed into E. coli ElectroMAXTM competent cells (Invitrogen-Thermo Fisher Scientific, Karlsruhe, Germany) using a Gene Pulser (Bio-Rad, Munich, Germany), 0.1 cm gap length cuvettes and the parameters 12.5 kV/cm, 200 and 25 F. Transformants were selected on Luria–Bertani agar plates containing 1 mg/L cefotaxime (Oxoid, Wesel, Germany). Isolates that were repeatedly negative in transformation experiments (repeated at least five times, using new DNA obtained by different methods, changing competent cells, electroporation machine and cuvettes) were considered potentially to have chromosomally mediated ESBLs and were studied further. The possible association of blaCTX-M genes with insertion sequences IS26 and ISEcp1 was investigated by PCR sequencing using the CTX-M-consensus and tnpIS26 primers [10], or ALA-3 [11] and a modified ALA4 (5 CTATCCGTACAAGGGAG 3 ), respectively. 2.2. Molecular typing and mapping of blaCTX-M genes Isolates were typed using pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA, as indicated in the PulseNet protocol (http://www.pulsenet-europe.org). Plasmid content was visualised by S1 nuclease PFGE [8]. To confirm the chromosomal location of blaCTX-M genes, genomic DNA was also analysed by PFGE of I-CeuI-digested DNA [4]. The XbaI, S1 and I-CeuI PFGE profiles were transferred onto nylon membranes and were then hybridised with blaCTX-M -specific probes [8], and additionally with a 16S rDNA probe (a 7.5-kb BamHI rrnB fragment from pKK3535) in the case of I-CeuI PFGE profiles. Isolates were assigned to different phylogenetic groups by a recently modified multiplex PCR [12] and were typed by multilocus sequence typing (MLST) (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Mating experiments using isolates ESBL-105, ESBL-723, ESBL746, ESBL-788 and ESBL-868 as donors (selected to represent different clones/enzymes) were carried out as previously described [8] on liquid broth (liquid mating) and agar (filter mating) at 22 ◦ C and 37 ◦ C using sodium-azide-resistant E. coli K12 J53
Table 1 Clinical Escherichia coli isolates analysed in this work. Isolate
ESBL
Other -lactamases
MLST
Phylogenetic group
XbaI PFGE a
Plasmids (kb)
Country
Isolation date
Origin
ESBL-91 ESBL-831 ESBL-884 ESBL-788 ESBL-105 ESBL-26 ESBL-35 ESBL-229 ESBL-723 ESBL-725 ESBL-772 ESBL-815 ESBL-72 ESBL-746 ESBL-787 ESBL-811 ESBL-868
CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-9 CTX-M-51 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15
– – TEM-1 TEM-1 – – TEM-1 OXA-1 OXA-1 TEM-1 TEM-1 TEM-1 – OXA-1; TEM-1 – TEM-1 OXA-1; TEM-1
ST38 ST38 ST38 ST3878b ST1266 ST38 ST156 ST648 ST38 ST131 ST2178 ST38 ST131 ST38 ST648 ST131 ST131
D D D B2 B2 D B1 D D B2 B1 D B2 D D B2 B2
X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17
145; 78 125 135 125; 82; 37 – 115 170 110 125; 110; 85 135; 60; 53; 30 105; 80 135; 50 105 115; 55 90 90 80
Netherlands UK UK UK Netherlands Netherlands Netherlands Germany UK UK UK UK Netherlands UK UK UK UK
2009 2009 2008 2008 2009 2009 2009 2009 2009 2009 2009 2008 2009 2009 2008 2009 2008
Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human
ESBL: extended-spectrum -lactamase; MLST: multilocus sequence typing; PFGE: pulsed-field gel electrophoresis. a Owing to the high variability of XbaI PFGE profiles found in the isolates, the profiles were named using X followed by a consecutive number. b ST3878 is a single locus variant of ST131, which shows a different icd allele (208).
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Fig. 1. Genomic macrorestriction of Escherichia coli isolates analysed in this work: (a) XbaI PFGE; (b) S1 PFGE; and (c) I-CeuI PFGE. Lane M, XbaI-digested DNA of Salmonella enterica serovar Braenderup H9812, used as size standard. The asterisks indicate bands that hybridised with the blaCTX-M probes. PFGE: pulsed-field gel electrophoresis.
(kindly provided by L. Martínez-Martínez) as the recipient strain. Transconjugants were selected on eosin–methylene blue agar plates (Oxoid) containing 1 mg/L cefotaxime and 100 mg/L sodium azide (Sigma–Aldrich, Hamburg, Germany).
3. Results and discussion Attempts to transform ESBL genes into a recipient strain failed repeatedly for only 17 (4.8%) of the 356 E. coli isolates and these
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were selected to investigate chromosomally encoded ESBLs. Of the 17 isolates, 11 produced CTX-M-15 enzyme, usually in addition to narrow-spectrum -lactamases such as TEM-1 (5 isolates), OXA-1 (2) or both (2) (Table 1). The remaining six isolates expressed CTXM group 9 ESBLs; four produced CTX-M-14 (with two co-producing TEM-1), whilst CTX-M-9 and CTX-M-51 ESBLs were detected in one isolate each (Table 1). With regard to the genetic environment of the blaCTX-M genes, as usually reported [8], in all cases the presence of ISEcp1 was detected 48 bp upstream of blaCTX-M-15 . However, no association with ISEcp1 or IS26 could be identified in CTX-M group 9-producing isolates. The 17 isolates belonged to phylogenetic groups D (9), B2 (6) and B1 (2) (Table 1). The isolates were assigned to seven different sequence types (STs) and generated a high variety of XbaI and I-CeuI PFGE and plasmid profiles (Table 1 and Fig. 1). Two MLST sequence types dominated: seven isolates (41%) from the UK and The Netherlands, all with different XbaI PFGE profiles, belonged to ST38 (phylogroup D); and four isolates (24%) from the UK and The Netherlands, also showing different XbaI PFGE profiles, belonged to ST131 (phylogroup B2). The two phylogroup B1 isolates were CTXM-15-producers with unique sequence types (ST156 and ST2178). All isolates except one (ESBL-105) contained plasmids, ranging from ca. 30 kb to ca. 170 kb in size (Table 1 and Fig. 1b). However, Southern hybridisation of S1 digests with blaCTX-M specific probes was negative for all, suggesting a chromosomal location for these genes. To confirm this, XbaI and I-CeuI PFGE profiles were hybridised with blaCTX-M -specific probes, and subsequently with 16S rDNA in the case of I-CeuI-profiles (Fig. 1a and c). The hybridisation results corroborated that all blaCTX-M genes were chromosomally located (Fig. 1). Moreover, mating experiments carried out with a subset of five isolates repeatedly failed to yield transconjugants. Strains ESBL-105 and ESBL815 showed two positive bands in the I-CeuI profiles in the hybridisation with the blaCTX-M -specific probe, suggesting the presence of two copies of the gene in their chromosomes (Fig. 1a and c). Interestingly, the chromosomal location of different blaCTX-M genes was detected only in human isolates (n = 17), most of them ascribed to phylogroups frequently linked to hospitals [4]. These isolates represented 12.9% of the human isolates (17/132) included within the SAFEFOODERA-ESBL collection subset (356 isolates from different sources including human, animal and food isolates) compared with 0/224 non-human isolates. Among the 356 isolates, only eight belonged to ST38, and only one that originated from poultry contained blaCTX-M-1 located on an IncI1 plasmid. The seven remaining isolates (genetically heterogeneous, and carrying CTX-M-14, -51 or -15) were those included in this work. The predominant chromosomal location of ESBL-encoding genes in ST38 isolates has already been observed in previous studies [13]. This interesting fact requires further investigation to elucidate whether these strains are really more prone to acquire these resistance genes in their chromosomes. On the other hand, 44/356 isolates (40 from humans, 3 from poultry and 1 of other origin) belonged to ST131 (the most frequent sequence type found in the 356 isolates, the second in frequency in the 17 isolates in this work), but only four of them had chromosomally mediated CTX-M enzymes and were included in this work. The rest of the ESBL-producing isolates of human, animal and food origin harboured the respective bla gene located on plasmids of different incompatibility groups (http://pubMLST.org; isolates submitted as ESBL-number; Woodford, unpublished data), as described by other authors [14]. Chromosomal integration of blaCTX-M genes has already been described in CTX-M-15- and CTX-M-14-producing E. coli clinical isolates from different European and Asian countries [4,5]. The strains in the current study, isolated in Germany, The Netherlands and the UK, showed a variety of sequence types and XbaI and I-CeuI
PFGE profiles. These facts may reveal that although the chromosomal location of blaCTX-M genes is less frequent than the plasmid location, this integrative phenomenon is not as uncommon as has been previously thought, and that spread of these strains is not due to the dispersion of a successful E. coli clone. This contrasts with the situation found in German E. coli isolated from livestock in which the increasing number of CTX-M-15-producing isolates is also associated both with isolates carrying IncI1 plasmids and with the spread of a ST410 clone that harbours the blaCTX-M-15 gene chromosomally (Guerra, personal communication). The spread of both blaCTX-M-15 and blaCTX-M-14 in human isolates is a combination of successful clones and plasmids. In this way, dissemination of CTX-M-15 in human isolates is classically attributed to the expansion of the E. coli O25b-ST131 clone harbouring IncF plasmids [2,3]. In the case of blaCTX-M-14 , however, its dissemination is mainly due to IncK plasmids, which constitute important vectors for the horizontal transfer of this gene to clonally unrelated E. coli isolates both from humans and animals [15]. In addition, ST38 is another major clone responsible for the spread of blaCTX-M-15 and blaCTX-M-14 in different continents of the world (http://mlst.warwick.ac.uk/mlst/) [5]. As described above, in the current series the predominance of these two sequence types (ST38 and ST131) both in blaCTX-M-15 - and blaCTX-M-14 -producing isolates was observed, and this is a remarkable fact that may indicate a significant role of these STs in this phenomenon. Currently the most successful multidrug-resistant (MDR) E. coli clones are predominantly associated with the production of CTXM enzymes. This is in contrast to Klebsiella pneumoniae, in which species clonal distribution has contributed to the occurrence of carbapenemases in hospitals, especially in southern European countries. This situation has led to the loss of control of these MDR pathogens in hospitals in countries such as Greece and Italy. The public health concern would be even higher if the carbapenemases also become widely distributed in the community in successful E. coli clones [1]. As far as we know, this is the first report describing the chromosomal location of blaCTX-M-9 and blaCTX-M-51 in E. coli isolates. Surveillance studies are essential to gain information about the occurrence and prevalence of these strains in different populations. More experimental work focused on the genetic environment of these chromosomal genes is also necessary to elucidate the genetic mechanism responsible for this integration as well as the exact location of the genes on the different chromosomes. Detection of ISEcp1 in some of the strains suggests the role of this insertion sequence in the chromosomal integration, either through transposition or homologous recombination. The presence of these antimicrobial resistance genes on the chromosome rather than on plasmids of different clinical E. coli isolates points to an evolutionary process that likely promotes the maintenance of the gene in the population. Funding: The Federal Institute for Risk Assessment (BfR) [BfR-45004; BfR-46-001] and the EU-SAFEFOODERA project EU ERA-Net, Ref. 08176, entitled ‘The role of commensal microflora of animals in the transmission of extended spectrum -lactamases’. During the experimental execution of this work, IR was a postdoctoral student at the BfR (Berlin, Germany), with a grant from the Fundación Ramón Areces (Madrid, Spain); she currently holds a postdoctoral position at Hospital Universitario Ramón y Cajal (Madrid, Spain). Competing interests: None declared. Ethical approval: Not required.
Acknowledgments The authors thank S. Schmoger, B. Baumann, W. Barownick and the NRL-Salm staff for their helpful assistance.
Please cite this article in press as: Rodríguez I, et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.02.019
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ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK I. Rodríguez a,∗ , K. Thomas a , A. Van Essen b , A.-K. Schink c , M. Day d , M. Chattaway d , G. Wu e , D. Mevius b,f , R. Helmuth a , B. Guerra a , on behalf of the SAFEFOODERA-ESBL consortium1 a
Federal Institute for Risk Assessment (BfR), Department for Biological Safety, Max-Dohrn Strasse 8–10, D-10589 Berlin, Germany Central Veterinary Institute (CVI) of Wageningen UR, Department of Bacteriology and TSEs, P.O. Box 65, 8200 AB Lelystad, The Netherlands c Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), 31535 Neustadt-Mariensee, Germany d Public Health England (PHE), 61 Colindale Avenue, London NW9 5EQ, UK e Animal Health and Veterinary Laboratories Agency (AHVLA), Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK f Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands b
a r t i c l e
i n f o
Article history: Received 21 October 2013 Accepted 26 February 2014 Keywords: Antimicrobial resistance CTX-M PFGE Plasmids
a b s t r a c t This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n = 356) obtained in Germany, The Netherlands and the UK (2005–2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. -Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 -lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Production of -lactamases is the main mechanism responsible for resistance to -lactams in Enterobacteriaceae. Extendedspectrum -lactamases (ESBLs) are able to hydrolyse third-
∗ Corresponding author. Present address: Department of Microbiology, Hospital Universitario Ramón y Cajal, Carretera de Colmenar Viejo Km 9.100, 28034 Madrid, Spain. Tel.: +34 913 368 152; fax: +34 913 368 809. E-mail address: [email protected] (I. Rodríguez). 1 This consortium also includes: Martin J. Woodward and Nick Coldham, Animal Health and Veterinary Laboratories Agency (AHVLA), UK; Kristina Kadlec and Stefan Schwarz, Friedrich-Loeffler-Institut (FLI), Germany; John Threlfall, Neil Woodford and John Wain, Public Health England (PHE), UK; and Cindy Dierikx, Central Veterinary Institute (CVI), The Netherlands.
and fourth-generation cephalosporins and monobactams, limiting therapeutic options in serious infections caused by Enterobacteriaceae. Over the last decade, the number of ESBL-producing bacteria has increased in many different genera of Enterobacteriaceae and represents a public health threat [1]. CTX-M-type ESBLs are a complex and heterogeneous family of enzymes and may be subdivided into five major groups (CTX-M1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 groups) [2]. These enzymes have spread globally and are the most common ESBLs detected in Enterobacteriaceae, not only in hospitals but also in the community, changing the epidemiology of ESBLs. Among the different CTX-M enzymes, CTX-M-15 (belonging to CTX-M group 1) and CTX-M-14 (belonging to CTX-M group 9) are of high relevance because of their ubiquity, being detected not only in humans and animals but also in environmental samples in many different
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Please cite this article in press as: Rodríguez I, et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.02.019
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countries [1,2]. The successful spread, diversification and maintenance of CTX-M enzymes is due to a combination of different factors: their association with transposable elements and integrons; the capture of these structures by conjugative plasmids; and their transfer to and maintenance in successful bacterial clones [2]. The latter are typified by Escherichia coli clone O25b-ST131, which is a pandemic, multiresistant and uropathogenic lineage frequently associated with the expression of CTX-M-15 and that has contributed significantly to the worldwide spread of this ESBL [3]. This clone habitually harbours the blaCTX-M-15 gene located on plasmids belonging to the IncF family, typically IncFII or multireplicon FII, FIA and FIB [3]. Despite being predominantly plasmid-mediated enzymes, chromosomally located blaCTX-M genes have also been described [4–6]. In this case, the chromosomal location does not enhance the spread of the gene but does assist its stabilisation and maintenance in the bacterium. The objective of this study was to seek and characterise clinical E. coli isolates suspected of expressing chromosomally encoded CTX-M enzymes from Germany, The Netherlands and the UK. 2. Methods 2.1. Bacterial isolates and detection of ˇ-lactamase-encoding genes Within the European SAFEFOODERA-ESBL project (EU ERANet, Ref. 08176), a total of 629 E. coli isolates were selected from the strain collections of the Animal Health and Veterinary Laboratories Agency (AHVLA, UK), Public Health England (PHE, UK), Central Veterinary Institute (CVI, The Netherlands), Friedrich-Loeffler-Institut (FLI, Germany) and Federal Institute for Risk Assessment (BfR, Germany). All isolates were cefotaximenon-susceptible [minimum inhibitory concentrations above the epidemiological cut-off of ≤0.25 mg/L for E. coli, set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); http://www.eucast.org]. Isolates originated from animals (n = 295), animal-derived foods (n = 59), humans (n = 274) and an unknown source (n = 1) and were isolated between 2005 and 2009. These isolates were screened for -lactamase and other resistance genes as well as for virulence determinants using ‘Amr05’ microarrays (ALERE Technologies Ltd., Stirling, UK) in a previous study [7]. For the present work, a subset of 356 isolates was selected
based on the relative contribution of the -lactamase families detected, the source of the isolates and the countries involved. Lactamase genes detected in these isolates with the array were further analysed by PCR sequencing as previously described [8,9]. Plasmid DNA was extracted from the 356 isolates using a Midiprep Plasmid Purification Kit (QIAGEN, Hilden, Germany) and/or the Kado and Liu method [8]. Plasmids were electrotransformed into E. coli ElectroMAXTM competent cells (Invitrogen-Thermo Fisher Scientific, Karlsruhe, Germany) using a Gene Pulser (Bio-Rad, Munich, Germany), 0.1 cm gap length cuvettes and the parameters 12.5 kV/cm, 200 and 25 F. Transformants were selected on Luria–Bertani agar plates containing 1 mg/L cefotaxime (Oxoid, Wesel, Germany). Isolates that were repeatedly negative in transformation experiments (repeated at least five times, using new DNA obtained by different methods, changing competent cells, electroporation machine and cuvettes) were considered potentially to have chromosomally mediated ESBLs and were studied further. The possible association of blaCTX-M genes with insertion sequences IS26 and ISEcp1 was investigated by PCR sequencing using the CTX-M-consensus and tnpIS26 primers [10], or ALA-3 [11] and a modified ALA4 (5 CTATCCGTACAAGGGAG 3 ), respectively. 2.2. Molecular typing and mapping of blaCTX-M genes Isolates were typed using pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA, as indicated in the PulseNet protocol (http://www.pulsenet-europe.org). Plasmid content was visualised by S1 nuclease PFGE [8]. To confirm the chromosomal location of blaCTX-M genes, genomic DNA was also analysed by PFGE of I-CeuI-digested DNA [4]. The XbaI, S1 and I-CeuI PFGE profiles were transferred onto nylon membranes and were then hybridised with blaCTX-M -specific probes [8], and additionally with a 16S rDNA probe (a 7.5-kb BamHI rrnB fragment from pKK3535) in the case of I-CeuI PFGE profiles. Isolates were assigned to different phylogenetic groups by a recently modified multiplex PCR [12] and were typed by multilocus sequence typing (MLST) (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Mating experiments using isolates ESBL-105, ESBL-723, ESBL746, ESBL-788 and ESBL-868 as donors (selected to represent different clones/enzymes) were carried out as previously described [8] on liquid broth (liquid mating) and agar (filter mating) at 22 ◦ C and 37 ◦ C using sodium-azide-resistant E. coli K12 J53
Table 1 Clinical Escherichia coli isolates analysed in this work. Isolate
ESBL
Other -lactamases
MLST
Phylogenetic group
XbaI PFGE a
Plasmids (kb)
Country
Isolation date
Origin
ESBL-91 ESBL-831 ESBL-884 ESBL-788 ESBL-105 ESBL-26 ESBL-35 ESBL-229 ESBL-723 ESBL-725 ESBL-772 ESBL-815 ESBL-72 ESBL-746 ESBL-787 ESBL-811 ESBL-868
CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-9 CTX-M-51 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15
– – TEM-1 TEM-1 – – TEM-1 OXA-1 OXA-1 TEM-1 TEM-1 TEM-1 – OXA-1; TEM-1 – TEM-1 OXA-1; TEM-1
ST38 ST38 ST38 ST3878b ST1266 ST38 ST156 ST648 ST38 ST131 ST2178 ST38 ST131 ST38 ST648 ST131 ST131
D D D B2 B2 D B1 D D B2 B1 D B2 D D B2 B2
X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17
145; 78 125 135 125; 82; 37 – 115 170 110 125; 110; 85 135; 60; 53; 30 105; 80 135; 50 105 115; 55 90 90 80
Netherlands UK UK UK Netherlands Netherlands Netherlands Germany UK UK UK UK Netherlands UK UK UK UK
2009 2009 2008 2008 2009 2009 2009 2009 2009 2009 2009 2008 2009 2009 2008 2009 2008
Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human
ESBL: extended-spectrum -lactamase; MLST: multilocus sequence typing; PFGE: pulsed-field gel electrophoresis. a Owing to the high variability of XbaI PFGE profiles found in the isolates, the profiles were named using X followed by a consecutive number. b ST3878 is a single locus variant of ST131, which shows a different icd allele (208).
Please cite this article in press as: Rodríguez I, et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.02.019
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Fig. 1. Genomic macrorestriction of Escherichia coli isolates analysed in this work: (a) XbaI PFGE; (b) S1 PFGE; and (c) I-CeuI PFGE. Lane M, XbaI-digested DNA of Salmonella enterica serovar Braenderup H9812, used as size standard. The asterisks indicate bands that hybridised with the blaCTX-M probes. PFGE: pulsed-field gel electrophoresis.
(kindly provided by L. Martínez-Martínez) as the recipient strain. Transconjugants were selected on eosin–methylene blue agar plates (Oxoid) containing 1 mg/L cefotaxime and 100 mg/L sodium azide (Sigma–Aldrich, Hamburg, Germany).
3. Results and discussion Attempts to transform ESBL genes into a recipient strain failed repeatedly for only 17 (4.8%) of the 356 E. coli isolates and these
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were selected to investigate chromosomally encoded ESBLs. Of the 17 isolates, 11 produced CTX-M-15 enzyme, usually in addition to narrow-spectrum -lactamases such as TEM-1 (5 isolates), OXA-1 (2) or both (2) (Table 1). The remaining six isolates expressed CTXM group 9 ESBLs; four produced CTX-M-14 (with two co-producing TEM-1), whilst CTX-M-9 and CTX-M-51 ESBLs were detected in one isolate each (Table 1). With regard to the genetic environment of the blaCTX-M genes, as usually reported [8], in all cases the presence of ISEcp1 was detected 48 bp upstream of blaCTX-M-15 . However, no association with ISEcp1 or IS26 could be identified in CTX-M group 9-producing isolates. The 17 isolates belonged to phylogenetic groups D (9), B2 (6) and B1 (2) (Table 1). The isolates were assigned to seven different sequence types (STs) and generated a high variety of XbaI and I-CeuI PFGE and plasmid profiles (Table 1 and Fig. 1). Two MLST sequence types dominated: seven isolates (41%) from the UK and The Netherlands, all with different XbaI PFGE profiles, belonged to ST38 (phylogroup D); and four isolates (24%) from the UK and The Netherlands, also showing different XbaI PFGE profiles, belonged to ST131 (phylogroup B2). The two phylogroup B1 isolates were CTXM-15-producers with unique sequence types (ST156 and ST2178). All isolates except one (ESBL-105) contained plasmids, ranging from ca. 30 kb to ca. 170 kb in size (Table 1 and Fig. 1b). However, Southern hybridisation of S1 digests with blaCTX-M specific probes was negative for all, suggesting a chromosomal location for these genes. To confirm this, XbaI and I-CeuI PFGE profiles were hybridised with blaCTX-M -specific probes, and subsequently with 16S rDNA in the case of I-CeuI-profiles (Fig. 1a and c). The hybridisation results corroborated that all blaCTX-M genes were chromosomally located (Fig. 1). Moreover, mating experiments carried out with a subset of five isolates repeatedly failed to yield transconjugants. Strains ESBL-105 and ESBL815 showed two positive bands in the I-CeuI profiles in the hybridisation with the blaCTX-M -specific probe, suggesting the presence of two copies of the gene in their chromosomes (Fig. 1a and c). Interestingly, the chromosomal location of different blaCTX-M genes was detected only in human isolates (n = 17), most of them ascribed to phylogroups frequently linked to hospitals [4]. These isolates represented 12.9% of the human isolates (17/132) included within the SAFEFOODERA-ESBL collection subset (356 isolates from different sources including human, animal and food isolates) compared with 0/224 non-human isolates. Among the 356 isolates, only eight belonged to ST38, and only one that originated from poultry contained blaCTX-M-1 located on an IncI1 plasmid. The seven remaining isolates (genetically heterogeneous, and carrying CTX-M-14, -51 or -15) were those included in this work. The predominant chromosomal location of ESBL-encoding genes in ST38 isolates has already been observed in previous studies [13]. This interesting fact requires further investigation to elucidate whether these strains are really more prone to acquire these resistance genes in their chromosomes. On the other hand, 44/356 isolates (40 from humans, 3 from poultry and 1 of other origin) belonged to ST131 (the most frequent sequence type found in the 356 isolates, the second in frequency in the 17 isolates in this work), but only four of them had chromosomally mediated CTX-M enzymes and were included in this work. The rest of the ESBL-producing isolates of human, animal and food origin harboured the respective bla gene located on plasmids of different incompatibility groups (http://pubMLST.org; isolates submitted as ESBL-number; Woodford, unpublished data), as described by other authors [14]. Chromosomal integration of blaCTX-M genes has already been described in CTX-M-15- and CTX-M-14-producing E. coli clinical isolates from different European and Asian countries [4,5]. The strains in the current study, isolated in Germany, The Netherlands and the UK, showed a variety of sequence types and XbaI and I-CeuI
PFGE profiles. These facts may reveal that although the chromosomal location of blaCTX-M genes is less frequent than the plasmid location, this integrative phenomenon is not as uncommon as has been previously thought, and that spread of these strains is not due to the dispersion of a successful E. coli clone. This contrasts with the situation found in German E. coli isolated from livestock in which the increasing number of CTX-M-15-producing isolates is also associated both with isolates carrying IncI1 plasmids and with the spread of a ST410 clone that harbours the blaCTX-M-15 gene chromosomally (Guerra, personal communication). The spread of both blaCTX-M-15 and blaCTX-M-14 in human isolates is a combination of successful clones and plasmids. In this way, dissemination of CTX-M-15 in human isolates is classically attributed to the expansion of the E. coli O25b-ST131 clone harbouring IncF plasmids [2,3]. In the case of blaCTX-M-14 , however, its dissemination is mainly due to IncK plasmids, which constitute important vectors for the horizontal transfer of this gene to clonally unrelated E. coli isolates both from humans and animals [15]. In addition, ST38 is another major clone responsible for the spread of blaCTX-M-15 and blaCTX-M-14 in different continents of the world (http://mlst.warwick.ac.uk/mlst/) [5]. As described above, in the current series the predominance of these two sequence types (ST38 and ST131) both in blaCTX-M-15 - and blaCTX-M-14 -producing isolates was observed, and this is a remarkable fact that may indicate a significant role of these STs in this phenomenon. Currently the most successful multidrug-resistant (MDR) E. coli clones are predominantly associated with the production of CTXM enzymes. This is in contrast to Klebsiella pneumoniae, in which species clonal distribution has contributed to the occurrence of carbapenemases in hospitals, especially in southern European countries. This situation has led to the loss of control of these MDR pathogens in hospitals in countries such as Greece and Italy. The public health concern would be even higher if the carbapenemases also become widely distributed in the community in successful E. coli clones [1]. As far as we know, this is the first report describing the chromosomal location of blaCTX-M-9 and blaCTX-M-51 in E. coli isolates. Surveillance studies are essential to gain information about the occurrence and prevalence of these strains in different populations. More experimental work focused on the genetic environment of these chromosomal genes is also necessary to elucidate the genetic mechanism responsible for this integration as well as the exact location of the genes on the different chromosomes. Detection of ISEcp1 in some of the strains suggests the role of this insertion sequence in the chromosomal integration, either through transposition or homologous recombination. The presence of these antimicrobial resistance genes on the chromosome rather than on plasmids of different clinical E. coli isolates points to an evolutionary process that likely promotes the maintenance of the gene in the population. Funding: The Federal Institute for Risk Assessment (BfR) [BfR-45004; BfR-46-001] and the EU-SAFEFOODERA project EU ERA-Net, Ref. 08176, entitled ‘The role of commensal microflora of animals in the transmission of extended spectrum -lactamases’. During the experimental execution of this work, IR was a postdoctoral student at the BfR (Berlin, Germany), with a grant from the Fundación Ramón Areces (Madrid, Spain); she currently holds a postdoctoral position at Hospital Universitario Ramón y Cajal (Madrid, Spain). Competing interests: None declared. Ethical approval: Not required.
Acknowledgments The authors thank S. Schmoger, B. Baumann, W. Barownick and the NRL-Salm staff for their helpful assistance.
Please cite this article in press as: Rodríguez I, et al. Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.02.019
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