L-015

CTX-M Producing Multi-drug Resistant Clinical Isolates of Escherichia coli in Norway are Associated with Virulent Genetic Lineages
Umaer Naseer , Bjrg Haldorsen , Kristin H Dahl , Flemming Scheutz , Gunnar S Simonsen , Timothy R Walsh , Arnfinn Sundsfjord and the Norwegian ESBL study group.
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Reference Centre for Detection of Antimicrobial Resistance (K-res), Department of Microbiology and Virology, IMB, University of Troms and Department of Microbiology and Infection Control, University Hospital of North-Norway, Troms, Norway 2 The International Escherichia and Klebsiella Centre (WHO) Statens Seruminstitutt, Copenhagen, Denmark and 3Department of Medical Microbiology, Cardiff, United Kingdom.
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Reference Centre for Detection of Antimicrobial Resistance Department of Microbiology and Infection Control University Hospital of Northern-Norway. N-9038 Troms, Norway. Umaer.Naseer@fagmed.uit.no Phone +47 776 45752 Fax +47 776 45350

Abstract

OBJECTIVE: Molecular typing of Norwegian clinical isolates of CTX-M producing Escherichia coli.

MATERIAL: E. coli with reduced susceptibility to 3rd generation cephalosporins (MIC > 1 mg/L) from 18 laboratories were submitted to the Reference Centre in 2003. Among the submitted 87 E. coli isolates, fifty were detected as ESBL-producing of which 45 were blaCTX-M positive. METHODS: The strains were characterised by antimicrobial susceptibility testing (Etest and Vitek2), blaCTX-M sequencing, ISEcp1- and In60-PCRs as well as -glucuronidase-, verocytotoxin- and alpha-haemolysin production. BlaCTX-M plasmids were characterized by hybridisation of PFGE separated S1-nuclease-digested total DNA, and for transferability by filter mating. Clonality was examined by XbaI-PFGE, phylogenetic grouping (A, B1, B2, and D) as well as O:K:H serotyping. RESULTS: We detected 29 CTX-M group 1 isolates of which blaCTX-M-15 was the most prevalent (n=23), CTX-M group 9 (n=15) and CTX-M group 2 isolates (n=1). Eighteen CTX-M group 1 and 4 CTX-M group 9 isolates were linked to ISEcp1 and In60, respectively. A majority of strains displayed unrelated XbaI-PFGE patterns, with some regional similarities among hospital and community strains in 5 minor clusters. Transferable blaCTX-M plasmids of 100 - 200 kb were observed. A total of 32 (71%) strains belonged to virulent groups B2 and D. The dominant serotype was O102:K 20,23:H 6 (7/45). -glucuronidase-, vero cytotoxin-, or alpha-haemolysin production was detected in 41/45 (91%), 0/45, and 2/45 (4%) isolates, respectively. Multidrug resistance (resistance to 2 non-betalactams) was detected in 35/45 isolates (78%). CONCLUSIONS: BlaCTX-M-15 and blaCTX-M-9 are the predominant ESBL-genotypes among E. coli in Norway. They emerge multi-centred and in the community. Clusters of XbaI-PFGErelated isolates share similar plasmid patterns, associated mobile genetic elements, phylogenetic groups and serotypes. Results show that Norwegian CTX-M producing E. coli strains are part of several multidrug-resistant and virulent lineages associated with extraintestinal infections.

Conjugation Transferability was analysed for 15 isolates to rifampicin resistant, plasmid free E. coli donor (J53-2) by R-factor transfer13. Transconjugants confirmed by XbaI- and S1-nuclease PFGE, and antimicrobial susceptibility profiling. Phylogenetic groups Phylogenetic relationship of the CTX-M producing E. coli isolates was investigated by triplex PCR, run for DNA fragments chuA, yjaA and TspE4.C2 (GeneAmp PCR System9700, Applied Biosystems)9. Serotyping Isolates were serotyped by SSI diagnostic antisera16 and analysed for the production of verocytotoxin using a Vero cell assay (VCA) and haemolysin on blood agar plates using 5% defibrinated washed sheep blood3 (Performed at the International Escherichia and Klebsiella Centre in Copenhagen).

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Ref# K4-81 K5-65 K4-69 K5-14 K5-01 K5-16 K9-61 K4-76 K5-62 K8-4 K5-59 K5-34 K5-43 K4-41 K4-23 K5-51 K8-1 K5-80 K4-47 K2-64 K4-40 K8-9 K5-56 K4-45 K5-12 K5-11 K2-70 K5-09 K5-58 K2-63 K4-55 K4-51 K5-60 K5-08 K5-64 K4-70 K2-55 K4-74 K5-76 K8-8 K4-31 K4-46 K4-79 A2-62 A2-63 K4-60 Sample CTX-M Genotype Urine Other Urine 15 Urine Other Other 1 Skin 1 Skin 15 Urine 15 Respiratory 15 Urine 15 Urine 15 Urine 15 Urine 15 Urine 15 Urine 9a Urine 9a Respiratory 15 Intestinal 15 Urine Other Urine 9a Urine 9a Intestinal 15 Urine 15 Urine 15 Urine 15 Respiratory 15 Respiratory 15 Urine 15 Respiratory 15 Urine 3 Urine 15 Other 1 Urine Other Urine Other Urine Other Urine Other Other Other Other 15 Urine 15 Urine Other Urine 3 Other Other Urine 2 Urine Other Unknown 15 Unknown 15 Respiratory 15 CTX-M Group 9 1 9 1 1 1 1 1 1 1 1 1 1 9 9 1 1 9 9 9 1 1 1 1 1 1 1 1 1 1 1 9 9 9 9 9 1 1 9 1 9 2 9 1 1 1 Hospital of Isolation St. Olavs Hospital Trondheim Ullevl US Ullevl US Ullevl US Ullevl US AHUS SST Rogaland (outbreak reference) Rikshospitalet SST Rogaland SST Rogaland SST Rogaland Rikshospitalet Ullevl US SST Rogaland SST Rogaland Unknown St. Olavs Hospital Trondheim Sykehus Vestfold HF SH Asker & Brum HF Sykehus Vestfold HF Srland Sykehus HF Haukeland Haukeland Rikshospitalet Ullevl US Ullevl US Ullevl US Ullevl US UNN Sykehuset Vestfold HF Lab Klin. Mikrob. Molde SH. Mikrobio Molde St. Olavs Hospital Trondheim Lillehammer Ullevl US Srland Sykehus HF AHUS Nordlandssh. Bod St. Olavs Hospital Trondheim Sykehus stfold HF SH Asker & Brum HF Ullevl US UK (Woodford) UK (Woodford) Ullevl US

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In the past decade cefotaximase (CTX-M) enzymes have become the most prevalent extended spectrum -lactamases (ESBLs) worldwide7. The CTX-M family is plasmid-mediated and have evolved from chromosomal -lactamase genes of Kluyvera spp.12. The first CTX-M to be sequence typed and named was in Germany in 19902. To date 65 CTX-M sequence types have been identified14, divided into five clusters based on their amino acid identities (1, 2, 8, 9 and 25)5. CTX-M-encoding genes have commonly been located on plasmids from 7kb8 to 430kb11. These plasmids have been shown to carry genes for resistance to multiple other antibiotics and to be transmissible in vitro4. Different elements are involved in the mobilization of blaCTX-M genes, ISEcp1 or ISEcp1-like insertion sequences have been observed 42 to 266 bp upstream of ORFs encoding CTX-Ms in all of the CTX-M groups4. CTX-M-9and CTX-M-2-encoding genes have been observed in class 1 integrons, designated InS2110, In351, and In6018. Most of the CTX-M enzymes are found in E. coli, and their potential relationship to a specific phylogenetic group has been investigated, revealing relationships to phylogenetic groups B26 and D15, considered to be most virulent and multidrug-resistant respectively.

Introduction

Results
Table 1 Distrubution of blaCTX-M genes in plasmids, their linkage to genetic elements and co-resistance profiles CTX-M Group (n) blaCTX-M (n) CTX-M-1 (4) 1 (29) CTX-M-3 (2) CTX-M-15 (23) 2 (1) 9 (15) CTX-M-2 (1) CTX-M-9/9a (4) Other (11) Size of blaCTX-M plasmid >200kb 2 1 1 1 200kb 2 3 150kb 17 100kb 2 8 50kb 3 1 2 Genetic Elements ISEcp1 1 1 16 nd nd nd In60 nd nd nd nd 4 CIP 1 2 20 1 7 Co-Resistance NIT 14 1 4 SXT 1 2 18 3 7 AG 1 21 3 3 Multi-Resistance 2 Other Drugs 1 2 22 3 8

Figure 2. Triplex PCR of 13 CTX-M isolates for the identification of phylogenetic relationship. Phylogenetic relationship is determined by fragment pattern of chuA (279bp), yjaA (211bp) and TspE4c2 (152bp) from a dichotomous decision tree described by Clermont et al.9. Lane content: 1; Kb ladder, 2; control pA, 3; control pB1, 4; control pB2, 5; control pD, 6; K2-55 (CTX-M-15), 7; K2-64 (CTX-M-9/9a), 8; K2-70 (CTX-M-15), 9; K4-23 (CTX-M-9/9a), 10; K4-31 (CTX-M-9), 11; K4-41 (CTX-M-9/9a), 12; K4-45 (CTX-M-15), 13; K4-46 (CTX-M-2), 14; K4-47 (CTX-M-9/9a), 15; K4-51 (CTX-M-9), 16; K4-55 (CTX-M-1), 17; K4-60 (CTX-M-15), 18; negative control, 19; Kb ladder.

Table 2 Linkage between Norwegian CTX-M producing E. coli clusters, multi-drug resistance, virulent phylogenetic relationship and serotype Reference # K5-56 K8-9 K5-11 K5-12 K2-70 K4-76 K5-34 K8-4 K5-59 K5-62 K4-23 K4-41 K5-14 K5-01 Genotype 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-9/9a CTX-M-9/9a CTX-M-1 CTX-M-1 XbaI Cluster A A B B B C C C C C D D E E BlaCTX-M Plasmid ~ 150 kb 150 kb 150 kb 150 kb >200 kb 150 kb 150 kb 150 kb 150 kb 150 kb 200 kb 200 kb 50 kb 50 kb Gen. element Linkage ISEcp1 ISEcp1 ISEcp1 ISEcp1 ISEcp1 ISEcp1 ISEcp1 In60 In60 CIP R R R R R R R R R R S S S S Multi-Drug Profile NIT R R S S R R R R R R S S S S SXT R R S R S R R R R R S S S S AG R R R R S R R R R R R R S S Phylogenetic Relationship B2 B2 B2 B2 B2 D D D D D D D B2 B2 Serotype O 25:K+:H 4 O 25:K 5:H 4 O 25:K100:H 4 O 25:K100:H 4 O 25:K-:H 4 O102:K 20,23:H 6 O102:K 20,23:H 6 O102:K 20,23:H 6 O102:K 20,23:H 6 O102:K 20,23:H 6 O 86:K+:H18 O 86:K+:H18 O 4:K 3:H 5 O 4:K 3:H 5 Serotyping PGUA + + + + + + + + + + + + + + HLY + VCA -

CIP: Ciprofloxacin NIT: Nitrofurantoin SXT: Trimetoprim-Sulphamethoxazole AG: Amino Glycosides nd: not determined

Molecular typing of Norwegian clinical isolates of CTX-M producing Escherichia coli.

Objective

BlaCTX-M distribution The 45 isolates were distributed into 29 CTX-M group 1 isolates, 15 group 9 isolates and 1 group 2 isolate (table 1). Antimicrobial susceptibility testing A total of 40 isolates (89%) displayed some level of co-resistance. Multi-resistant (resistant to 2 other classes of antibiotics) was observed in 35 (78%) isolates (table 1). Genetic environment ISEcp1 was detected in 18 (62%) of the group 1 isolates and In60 was detected in 4 (27%) of the CTX-M group 9 isolates (table 1). Chromosomal typing XbaI-PFGE patterns were obtained for 43 (95.5%) isolates representing 36 different pulsed field-types including 5 clusters of related isolates (>85% similarity), labelled A-E (figure 1). DNA from two isolates consistently autodigested and no PFGE-patterns were obtained. Plasmid typing S1-nuclease plasmid profiles were obtained for 43 (96 %) isolates contained 1 to 3 plasmids from ~20 to 200 kb, high molecular plasmids of >200 kb were not separated. Successful hybridisation identified blaCTX-M plasmids (table 1)
References

Material 45 clinical isolates of E. coli with confirmed CTX-M production19, from 18 Norwegian diagnostics laboratories in 200319. Antimicrobial susceptibility testing Performed using agar disc diffusion (Oxoid, Basington, UK), Etest (AB Biodisk, Solna Sweden) and VITEK2 (bioMerieux). Genetic environment ISEcp1 and In60 RFLP-PCRs were performed on CTX-M groups 1 and 9 isolates respectively, using restriction enzymes PvuII and NotI, (New England, BioLabs, Beverly USA) and a standard PCR reaction mix with GeneAmp PCR buffer and Taq DNA polymerase (Applied Biosystems). Chormosomal typing Performed using pulsed field gel electrophoresis (PFGE) typing of XbaI digested DNA, run on a multidirectional gel electrophoresis, provided by Chef-DR III System (BIO-RAD, Oslo, Norway). Plasmid typing Performed using S1-nuclease digestion (linearisation) of circular dsDNA plasmids run on a PFGE, provided by Chef-DR III System (BIO-RAD, Oslo, Norway). BlaCTX-M plasmid identification was performed Southern hybridisation (Roche Diagnostics GmbH, Mannheim, Germany) with CTX-M specific probes (PCR DIG Probe Synthesis Kit QIAGEN, Hilden, Germany17).

Methods

Figure 1. Dendrogram to illustrate the genetic relatedness of 43 CTX-M-producing E. coli isolates from Norway examined by PFGE. The strain reference number (Ref #), type of clinical sample (Sample), CTX-M genotype and group (CTX-M- genotype, group) as well as the laboratory of isolation (Hospital of isolation) is given for each strain. Major strains defined by PFGE profiles with 85% similarity (black vertical line), are indicated. Each gradation on the scale represents a 5% difference in similarity. A total of 31 single types are identified and five cluster types of genetically related strains are labelled A-E, encircled in red. PFGE patterns were not obtained from two isolates due to DNA autodigestion.

CIP: Ciprofloxacin NIT: Nitrofurantoin SXT: Trimetoprim-Sulphamethoxazole AG: Amino Glycosides R: Resistant S: Sensitive PGUA: -glucuronidase HLY: -hemolysin VCA: Vero cytotoxin

Conjugation R-factor transfer of blaCTX-M was achieved for 8 (53%) isolates with transformation frequencies ranging from 1.6 x10-7 to 7.8 x10-4. Co-transfer of resistance was only observed for trimetoprim sulphamethoxazole in 3 out of 7 isolates (43 %). Phylogenetic groups Isolates were distributed according to phylogenetic classification into the virulent phylogenetic groups B2 13 (29%) and D 20 (44%), and the commensal groups A 9 (20%), B1 3 (7%) (figure 2). Serotyping Isolates expressed a wide variety of different O:K:H serotypes. The dominant serotype O102: K20, 23:H6 was expressed in 7 (16%) isolates. No vero cytotoxin production was detected; -glucuronidase production was detected in 41 (91%) and alpha-hemolysin in 2 (4%) isolates.

1. The emergence of CTX-M producing E. coli in Norway appears to be multi-centred (figure 1). 2. High degree of PFGE heterogeneity was observed among the isolates with a few clusters of related isolates (figure 1). 3. BlaCTX-Ms were identified on large transferable plasmids (> 100kb) with the ability to co-transfer trimetoprim sulphamethoxazole resistance. 4. CTX-M-15 was the most dominant genotype and linked to ISEcp1 in 70% of the isolates (table 1). 5. BlaCTX-M isolates were shown to be multi-drug resistant (78%) (table1). 6. BlaCTX-M isolates were identified as part of virulent genetic lineages of E. coli classified into phylogenetic groups B2 and D (figure 2). 7. We observed a strong concordance between different phenotypic (serotype, antibiotic profile) and genetic (PFGE, phlogeny) typing methods (table 2).
Design/Layout: Rod Wolstenholme, AV Dept., The Medical Faculty, University of Troms, Norway

Conclusions

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Arduino, S. M., P. H. Roy, G. A. Jacoby, B. E. Orman, S. A. Pineiro, and D. Centron. 2002. blaCTX-M-2 is located in an unusual class 1 integron (In35) which includes Orf513. Antimicrob.Agents Chemother. 46:2303-2306. Bauernfeind, A., H. Grimm, and S. Schweighart. 1990. A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection 18:294-298. Beutin, L., M. A. Montenegro, I. Orskov, F. Orskov, J. Prada, S. Zimmermann, and R. Stephan. 1989. Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J.Clin.Microbiol. 27:2559-2564. Bonnet, R. 2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob.Agents Chemother. 48:1-14. Bradford, P. A. 2001. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin.Microbiol.Rev. 14:933-51, table. Branger, C., O. Zamfir, S. Geoffroy, G. Laurans, G. Arlet, H. V. Thien, S. Gouriou, B. Picard, and E. Denamur. 2005. Genetic background of Escherichia coli and extended-spectrum beta-lactamase type. Emerg.Infect.Dis. 11:54-61. Canton, R. and T. M. Coque. 2006. The CTX-M beta-lactamase pandemic. Curr.Opin.Microbiol. 9:466-475. Cao, V., T. Lambert, and P. Courvalin. 2002. ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum beta-lactamase CTX-M-17. Antimicrob.Agents Chemother. 46:1212-1217. Clermont, O., S. Bonacorsi, and E. Bingen. 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl.Environ.Microbiol. 66:4555-4558. Di, C. J., J. A. Ayala, P. Power, M. Mollerach, and G. Gutkind. 2002. Novel class 1 integron (InS21) carrying blaCTX-M-2 in Salmonella enterica serovar infantis. Antimicrob.Agents Chemother. 46:2257-2261. Garcia, A., F. Navarro, E. Miro, B. Mirelis, S. Campoy, and P. Coll. 2005. Characterization of the highly variable region surrounding the blaCTX-M-9 gene in non-related Escherichia coli from Barcelona. J.Antimicrob.Chemother.

12. 13. 14. 15. 16. 17. 18. 19.

Gazouli, M., N. J. Legakis, and L. S. Tzouvelekis. 1998. Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A beta-lactamase CTX-M-4. FEMS Microbiol.Lett. 169:289-293. Kristin H.Dahl, Anne-Merethe Hanssen, and Johanna Ericson Sollid. R-plasmid transfer in Gram-negative Enterobacteriaceae. 2006. Ref Type: Data File Lahey. http://www.lahey.org/Studies/?D=http://www.lahey.org/studies/webt.htm&C=404. 31-10-2005. Ref Type: Data File Machado, E., R. Canton, F. Baquero, J. C. Galan, A. Rollan, L. Peixe, and T. M. Coque. 2005. Integron content of extended-spectrum-beta-lactamase-producing Escherichia coli strains over 12 years in a single hospital in Madrid, Spain. Antimicrob.Agents Chemother. 49:1823-1829. Orskov, F. and I. Orskov. 1984. Serotyping of Escherichia coli. Methods in microbiology 14.:43-112. QIAGEN. QIAquick Spin Handbook. 2005. Ref Type: Data File Sabate, M., F. Navarro, E. Miro, S. Campoy, B. Mirelis, J. Barbe, and G. Prats. 2002. Novel complex sul1-type integron in Escherichia coli carrying bla(CTX-M-9). Antimicrob.Agents Chemother. 46:2656-2661. Tofteland, S., B. Haldorsen, K. H. Dahl, G. S. Simonsen, M. Steinbakk, T. R. Walsh, and A. Sundsfjord. 2006. Extended spectrum -lactamase producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway: phenotypes, genotypes and consequences for detection methods. J.Clin.Microbiol.