JAC Advance Access originally published online on March 11, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1024-1028; doi:10.1093/jac/dkn084
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
The CTX-M-15-producing Escherichia coli diffusing clone belongs to a highly virulent B2 phylogenetic subgroup
1 INSERM U722 and Université Denis Diderot-Paris 7, Faculté de Médecine, Site Xavier Bichat, Paris, France 2 Université Pierre et Marie Curie-Paris 6, Faculté de Médecine, Laboratoire de Bactériologie, EA 2392 Paris, France 3 Assistance Publique-Hôpitaux de Paris, Hôpital Tenon, Service de Bactériologie-Hygiène, Paris, France 4 Assistance Publique-Hôpitaux de Paris, Hôpital Louis Mourier, Laboratoire de Microbiologie, Colombes, France 5 Université d'Auvergne, Faculté de Pharmacie, Laboratoire de Bactériologie, Clermont-Ferrand, France
* Correspondence address. Laboratoire de Bactériologie, Hôpital Tenon, 4 rue de la Chine, 75970 Paris Cedex 20, France. Tel: +33-1-56-01-70-18; Fax: +33-1-56-01-61-08; E-mail: guillaume.arlet{at}tnn.aphp.fr
Received 16 December 2007; returned 9 January 2008; revised 6 February 2008; accepted 8 February 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Objectives: A clone of CTX-M-15-producing Escherichia coli has recently been reported to be spreading through Europe and Africa. The aim of this work was to thoroughly characterize this clone.
Materials and methods: Representative isolates of this clone were subjected to multilocus sequence typing, O typing, virulence gene detection, adhesion assay on human cells, biofilm production assay and mouse lethality assay.
Results: The clone: (i) belongs to a unique B2 phylogenetic subgroup encompassing the pyelonephritogenic diffusely adhering EC7372 strain; (ii) exhibits a specific O25b molecular subtype; (iii) is identical to the E. coli clone O25:H4-ST131 producing CTX-M-15; (iv) produces biofilm; and (v) is highly virulent in mice despite lacking classical extraintestinal pathogenicity islands (except for high pathogenicity island) and the afa/dra gene.
Conclusions: The CTX-M-15-producing E. coli diffusing clone is associated with a high level of antibiotic resistance and with high virulence, showing that, under certain selective pressures, the previously observed trade-off between resistance and virulence may not apply.
Keywords: E. coli , ESBLs , virulence , clonal spread , B2 phylogenetic group
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Phylogenetic analyses show that Escherichia coli strains fall into four main phylogenetic groups (A, B1, B2 and D).1 Although virulence determinants are considered to be mobile, a link between strain phylogeny and virulence has been reported.1 Virulent extraintestinal strains belong mainly to B2 group and, to a lesser extent, D group, whereas most commensal strains belong to A and B1 groups.1 The prevalence of antimicrobial resistance is lower among strains belonging to B2 phylogenetic group, suggesting a trade-off between resistance and virulence.2
In recent years, the epidemiology of extended-spectrum β-lactamases (ESBLs) in E. coli has changed radically: (i) novel ESBLs such as CTX-M enzymes have replaced classical TEM and SHV-type ESBLs in many countries3 and (ii) infections by these ESBL-producing E. coli are described both in hospitals and in the community.3 CTX-M-15 β-lactamase, which belongs to the CTX-M-1 group, was first described in 2001, but is now found worldwide in E. coli strains, notably during outbreaks in Canada, France, the UK, Spain and Tunisia.3 We showed the spread of a CTX-M-15 β-lactamase-producing E. coli clone in Paris, Tunis and Bangui.4
To better understand the causes of the evolutionary success of the CTX-M-15 β-lactamase-producing E. coli diffusing clone, we reconstructed its phylogenetic history and characterized its virulence in vitro and in vivo.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
E. coli strains
Four representative isolates of the multiresistant CTX-M-15-producing E. coli diffusing clone were selected from the panel used in our previous work (Table 1).4 Two were recovered from Tenon hospital in Paris (TN03 and TN34), and the other two were isolated in Tunis, Tunisia (strain TU) and in Bangui, Central African Republic (strain CAF).4 One isolate from France [TNN (TE2)] of the recently published CTX-M-15 clone belonging to the ST131 using the Achtman schema was also used for comparison.5 For the multilocus sequence typing (MLST) analysis, 21 B2 and 1 D strains as well as Escherichia fergusonii were used.6 In vitro and in vivo experiments were performed on a subset of the above-cited strains. In addition, the TN03 transformant in E. coli DH10B (EpTN03) producing the CTX-M-15 enzyme was used as a control in the biofilm experiment.
|
Phylogenetic grouping and MLST
The phylogenetic group of the E. coli isolates was determined with the multiplex PCR-based method.7 MLST was achieved by analysis of the concatenated sequences (5901 bp) of six essential genes (trpA, trpB, pabB, putP, icd and polB), with E. fergusonii as the outgroup, and processed by the maximum likelihood method in the PHYML program.7
The isolates were screened for 21 genes encoding putative virulence factors [neuC (K1 antigen), sfa/foc, iroN, iutA, iha, papC, papG (I, II, and III alleles), hlyC, cnf1, hra, sat, ire, usp, ompT, ibeA, malX, fyuA, irp2, traT, afa/dra and ftr1] by PCR with primers as previously described.7 Primers for ftr1, ftr1up (5'-GATCAGCGCCACAATTAC-3') and ftr1low (5'-CAGTGGAGTCTGCTGCTC-3'), were used at an annealing temperature of 55°C to generate a PCR product of 153 bp.
O typing was performed with the traditional antiserum method at the State Serum Institute, Copenhagen, Denmark. The O types were also determined with a recently described molecular approach based on allele-specific PCR.8 For the newly described O25b O type, primers rfb.1bis (5'-ATACCGACGACGCCGATCTG-3') and rfbO25b.r (5'-TGCTATTCATTATGCGCAGC-3') were used at an annealing temperature of 60°C to generate a PCR product of 300 bp. To define the rfbO25b.r primer, the entire rfb cluster was amplified and the 5' extremity was sequenced as previously described in strains ECOR15, EC7372 and TN03.8
Human intestinal cell adhesion assays
Adhesion to Int 407 cells, derived from a human embryonic jejunum and ileum, was assayed as previously described.9 Three independent adhesion assays were used for each strain.
Biofilm formation was examined twice in 60 mL aerated microfermentors, as previously described.10
A mouse model of systemic infection was used to assess the intrinsic virulence of the strains.1 For each strain, 10 outbred female Swiss OF1 mice (3–4 weeks old, 14–18 g) received a subcutaneous abdominal injection of 109 cfu/mL of log-phase bacteria. Mortality was monitored for 7 days.1
Animals were maintained and handled according to the guidelines of the French Ministry of Agriculture (approval A 75-18-05).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
The four CTX-M-15 isolates (TN03, TN34, TU and CAF) belong to phylogenetic group B2 (Table 1) and are grouped together by MLST with the pyelonephritogenic diffusely adhering EC7372 reference strain,6 with a high bootstrap value (Figure 1). These five strains are closely related to the diarrhoeic diffusely adhering DAEC11 and DAEC18 strains,6 and, to a lesser extent, to the ECOR66 strain (Figure 1). All these strains belong to the B2 phylogenetic subgroup I, which is the more basal (anciently diverged) strain in the B2 phylogenetic group.7
|
All four CTX-M-15 strains were O type O25 in the antiserum technique, but no PCR product was obtained with the O25-specific primer (rfbO25a) used in the recently published allele-specific method.8 In contrast, two control group B2 strains (ECOR51 and 52) belonging to subgroup II were successfully typed as O25 (O25a) by both approaches (Figure 1). Sequencing of the 5' extremity of the rfb cluster yielded an unknown sequence that we have already observed in strain ECOR15 (phylogenetic group A).8 We therefore designed a primer specific for this sequence (rfbO25b.r) and confirmed that the four CTX-M-15 isolates, EC7372, DAEC11, DAEC18 and ECOR15 strains, belong to this distinct O25 type (O25b) (Figure 1 and Table 1).
The four clonal isolates did not possess the classical extraintestinal PAIs (PAI ICFT073, PAI IIJ96 and PAI III536),7 except for the high pathogenicity island (HPI). They also lacked the afa/dra gene responsible for the diffusely adhering phenotype (Table 1). The four isolates had the same virulence pattern, except that isolate TN34 harboured iroN and isolate TU did not harbour ftr1. As the absence of sfa/foc in isolate TN34 indicates that iroN is plasmid-borne (Table 1), the differences in the virulence gene patterns between the clonal isolates are all due to plasmids.
The MLST analysis with our schema (Figure 1), the PCR-based O typing and virulence genotyping (Table 1) of strain TNN (TE2), a French isolate of the CTX-M-15 O25:H4-ST131 clone, show that our four isolates from France, Tunisia and Central African Republic and those spreading in France, Switzerland, Spain, Portugal, Lebanon, Korea and Canada belong to the same clone.5 Also, according to the serotype and/or the virulence genotype and/or the phylogenetic group, this worldwide diffusing clone is probably identical to those previously described in the UK, Madrid and Rome.3,11,12
As the TN03 strain is closely related to the diffusely adhering DAEC11 and DAEC18 strains, we examined the adhesion of these strains, and the ECOR66 strain, to intestinal cells. Similar to the ECOR66 strain, TN03 was less adherent than the DAEC11 and DAEC18 strains (Table 1). No specific pattern of adherence was observed with strains TN03 and ECOR66, whereas the DAEC strains showed typical diffuse adherence (data not shown), in keeping with their lack of afa/dra (Table 1).
We then examined whether TN03 and TN34 and the transformant EpTN03 could produce a biofilm on a glass support. TN03 and TN34 produced biofilm after 48 h, whereas EpTN03 produced none, showing that this property is not harboured by the plasmid encoding the blaCTX-M-15 gene; see Table 1 and Figure S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].
We also tested the four isolates of the CTX-M-15 clone in a mouse model of extraintestinal virulence.1 Despite the apparently small number of virulence genes, all CTX-M-15 isolates killed all 10 of the 10 inoculated mice, as the pyelonephritogenic antibiotic-susceptible EC7372 strain and ECOR66 strain exhibit several PAIs (Table 1). Interestingly, DAEC11 and DAEC18 also killed all 10 mice, indicating a high extraintestinal potential virulence within strains of subgroup I.
Why is the CTX-M-15-producing E. coli clone so successful? Usually, B2 phylogenetic group E. coli clinical isolates that do not produce ESBL belong rarely (~2%) to O subgroup O25b (Clermont and Denamur, personal data). In a recent study, O25 isolates were responsible for 38% of the upper urinary tract infection (UTI) in renal transplant patients and were associated with poor prognosis, whereas O25 strains represented <2% of the isolates from immunocompetent patients with upper UTI.13 These O25 strains possess P fimbriae papG class II and Dr adhesin, suggesting that they are similar to the EC7372 strain (Table 1). The authors suggested that a unique pattern of O type and adherence factors contributed to acute allograft injury in these renal transplant patients with UTI.13 Indeed, the EC7372 strain is highly virulent in the mouse model.7 Surprisingly, although closely related to strain EC7372, our clone did not produce these particular adhesion factors and did not adhere very avidly to epithelial intestinal cells (Table 1). The CTX-M-15 clone isolates produced biofilm: a property not supported by the CTX-M plasmid. This biofilm production could contribute to the long-term persistence of this clone in various environments and to its resistance to antimicrobial agents or disinfectants.10 Biofilms also confer increased resistance to host immune defences. Anderson et al.14 described the formation, in bladder cells, of intracellular bacterial communities that matured in biofilm-like structures (pods). Recently, it was reported that E. coli strains causing recurrent UTI were more likely to produce biofilms and to synthesize yersiniabactin (fyu) and aerobactin (aer/iutA), like our clone.15 The CTX-M-15 clone isolates were also highly virulent in the mouse model of extraintestinal virulence, even though they lack several classical virulence genes. This indicates that these isolates possess unidentified virulence genes and that these genes could have a role in clonal spread.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
This work was partially funded by a ‘Contrat d'Initiation à la Recherche Clinique’ CRC 05 103 (Assistance Publique, Hôpitaux de Paris), the ‘Fondation pour la Recherche Médicale’, the Faculté de Médecine Pierre et Marie Curie, Université Paris 6, and the European Community (6th PCRD Contract: LSHM-CT 2003-503335).
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Figure S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Footnotes |
|---|
O. Clermont and M. Lavollay contributed equally to this work. 
|
Acknowledgements |
|---|
We are grateful to Tony Le Gall for his help in the tree reconstruction and to Valérie Gautier for expert technical assistance.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
1 Picard B, Garcia JS, Gouriou S, et al. The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect Immun (1999) 67:546–53.
2
Johnson JR, Goullet P, Picard B, et al. Association of carboxylesterase B electrophoretic pattern with presence and expression of urovirulence factor determinants and antimicrobial resistance among strains of Escherichia coli that cause urosepsis. Infect Immun (1991) 59:2311–5.
3
Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother (2007) 59:165–74.
4
Lavollay M, Mamlouk K, Frank T, et al. Clonal dissemination of a CTX-M-15 β-lactamase-producing Escherichia coli strain in the Paris area, in Tunis and in Bangui. Antimicrob Agents Chemother (2006) 50:2433–8.
5
Nicolas-Chanoine MH, Blanco J, Leflon-Guibout V, et al. Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother (2008) 61:273–81.
6
Escobar-Páramo P, Clermont O, Blanc-Potard AB, et al. A specific background is required for acquisition and expression of virulence factors in Escherichia coli. Mol Biol Evol (2004) 21:1085–94.
7
Le Gall T, Clermont O, Gouriou S, et al. Extraintestinal virulence is a coincidental by product of commensalism in B2 phylogenetic group Escherichia coli strains. Mol Biol Evol (2007) 24:2373–84.
8 Clermont O, Johnson JR, Menard M, et al. Determination of Escherichia coli O types by allele-specific polymerase chain reaction: application to the O types involved in human septicemia. Diagn Microbiol Infect Dis (2006) 57:129–36.[Web of Science][Medline]
9
Favre-Bonte S, Joly B, Forestier C. Consequences of reduction of Klebsiella pneumoniae capsule expression on interactions of this bacterium with epithelial cells. Infect Immun (1999) 67:554–61.
10 Ghigo JM. Natural conjugative plasmids induce bacterial biofilm development. Nature (2001) 412:442–5.[CrossRef][Medline]
11
Karisik E, Ellington MJ, Livermore DM, et al. Virulence factors in Escherichia coli with CTX-M-15 and other extended-spectrum β-lactamases in the UK. J Antimicrob Chemother (2008) 61:54–8.
12
Carattoli A, Garcia-Fernandez A, Varesi P, et al. Molecular epidemiology of Escherichia coli producing extended-spectrum β-lactamases isolated in Rome, Italy. J Clin Microbiol (2008) 46:103–8.
13 Rice JC, Peng T, Kuo YF, et al. Renal allograft injury is associated with urinary tract infection caused by Escherichia coli bearing adherence factors. Am J Transplant (2006) 6:2375–83.[CrossRef][Web of Science][Medline]
14
Anderson GG, Palermo JJ, Schilling JD, et al. Intracellular bacterial biofilm-like pods in urinary tract infections. Science (2003) 301:105–7.
15 Soto SM, Smithson A, Horcajada JP, et al. Implication of biofilm formation in the persistence of urinary tract infection caused by uropathogenic Escherichia coli. Clin Microbiol Infect (2006) 12:1034–6.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Radhouani, L. Pinto, C. Coelho, A. Goncalves, R. Sargo, C. Torres, G. Igrejas, and P. Poeta Detection of Escherichia coli harbouring extended-spectrum {beta}-lactamases of the CTX-M classes in faecal samples of common buzzards (Buteo buteo) J. Antimicrob. Chemother., November 5, 2009; (2009) dkp403v1. [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. Sidjabat, D. L. Paterson, J. M. Adams-Haduch, L. Ewan, A. W. Pasculle, C. A. Muto, G.-B. Tian, and Y. Doi Molecular Epidemiology of CTX-M-Producing Escherichia coli Isolates at a Tertiary Medical Center in Western Pennsylvania Antimicrob. Agents Chemother., November 1, 2009; 53(11): 4733 - 4739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Johnson, S. Miller, B. Johnston, C. Clabots, and C. DebRoy Sharing of Escherichia coli Sequence Type ST131 and Other Multidrug-Resistant and Urovirulent E. coli Strains among Dogs and Cats within a Household J. Clin. Microbiol., November 1, 2009; 47(11): 3721 - 3725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Duval, I. Maiga, A. Maiga, T. Guillard, L. Brasme, D. Forte, J. Madoux, V. Vernet-Garnier, and C. De Champs High Prevalence of CTX-M-Type {beta}-Lactamases among Clinical Isolates of Enterobacteriaceae in Bamako, Mali Antimicrob. Agents Chemother., November 1, 2009; 53(11): 4957 - 4958. [Full Text] [PDF]
|
|
|
|
|
|
J. Oteo, B. Orden, V. Bautista, O. Cuevas, M. Arroyo, R. Martinez-Ruiz, M. Perez-Vazquez, M. Alcaraz, S. Garcia-Cobos, and J. Campos CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin J. Antimicrob. Chemother., October 1, 2009; 64(4): 712 - 717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Clermont, H. Dhanji, M. Upton, T. Gibreel, A. Fox, D. Boyd, M. R. Mulvey, P. Nordmann, E. Ruppe, J. L. Sarthou, et al. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains J. Antimicrob. Chemother., August 1, 2009; 64(2): 274 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Johnson, M. Menard, B. Johnston, M. A. Kuskowski, K. Nichol, and G. G. Zhanel Epidemic Clonal Groups of Escherichia coli as a Cause of Antimicrobial-Resistant Urinary Tract Infections in Canada, 2002 to 2004 Antimicrob. Agents Chemother., July 1, 2009; 53(7): 2733 - 2739. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Arpin, C. Quentin, F. Grobost, E. Cambau, J. Robert, V. Dubois, L. Coulange, C. Andre, and on behalf of the Scientific Committee of ONERBA Nationwide survey of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in the French community setting J. Antimicrob. Chemother., June 1, 2009; 63(6): 1205 - 1214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Blanco, M. P. Alonso, M.-H. Nicolas-Chanoine, G. Dahbi, A. Mora, J. E. Blanco, C. Lopez, P. Cortes, M. Llagostera, V. Leflon-Guibout, et al. Molecular epidemiology of Escherichia coli producing extended-spectrum {beta}-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15 J. Antimicrob. Chemother., June 1, 2009; 63(6): 1135 - 1141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Pomba, J. D. da Fonseca, B. C. Baptista, J. D. Correia, and L. Martinez-Martinez Detection of the Pandemic O25-ST131 Human Virulent Escherichia coli CTX-M-15-Producing Clone Harboring the qnrB2 and aac(6')-Ib-cr Genes in a Dog Antimicrob. Agents Chemother., January 1, 2009; 53(1): 327 - 328. [Full Text] [PDF]
|
|
|
|
|
|
S. H. Lau, M. E. Kaufmann, D. M. Livermore, N. Woodford, G. A. Willshaw, T. Cheasty, K. Stamper, S. Reddy, J. Cheesbrough, F. J. Bolton, et al. UK epidemic Escherichia coli strains A-E, with CTX-M-15 {beta}-lactamase, all belong to the international O25:H4-ST131 clone J. Antimicrob. Chemother., December 1, 2008; 62(6): 1241 - 1244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Leflon-Guibout, J. Blanco, K. Amaqdouf, A. Mora, L. Guize, and M.-H. Nicolas-Chanoine Absence of CTX-M Enzymes but High Prevalence of Clones, Including Clone ST131, among Fecal Escherichia coli Isolates from Healthy Subjects Living in the Area of Paris, France J. Clin. Microbiol., December 1, 2008; 46(12): 3900 - 3905. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||