JAC Advance Access published online on October 18, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn428
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Replicon typing of plasmids in Escherichia coli producing extended-spectrum β-lactamases
1 Université Pierre et Marie Curie-Paris-6, Faculté de Médecine, Site Saint-Antoine, Laboratoire de Bactériologie, EA 2392 Paris, France 2 Assistance Publique-Hôpitaux de Paris, Hôpital Tenon, Service de Bactériologie-Hygiène, Paris, France 3 INSERM U722 and Université Denis Diderot-Paris-7, Faculté de Médecine, Site Xavier Bichat, Paris, France 4 INSERM U707, Université Pierre et Marie Curie-Paris-6, Faculté de Médecine, Site Saint-Antoine, Paris, France 5 Assistance Publique-Hôpitaux de Paris, Hôpital Louis Mourier, Laboratoire de Microbiologie, Colombes, 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 3 May 2008; returned 23 June 2008; revised 20 September 2008; accepted 22 September 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Objectives: Escherichia coli producing CTX-M-15 and CTX-M-14 extended-spectrum β-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the replicons involved in the emergence and spread of ESBLs in relation to ESBL type.
Methods: A collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli strains was analysed. The replicons carrying the ESBLs and the total plasmid content of the strains have been characterized by PCR replicon typing in relation to the type of ESBL. The ESBL replicons were then compared with the replicon content of E. coli strains carrying TEM-1 or inhibitor-resistant TEM (IRT) β-lactamases.
Results: IncF plasmids were the most frequently carried replicons in our collection, but none carried TEM ESBL. Of TEM ESBLs, 67% were carried on IncA/C replicons except for TEM-52 genes, which were carried preferentially on IncI1 replicons. Although CTX-M enzymes can be carried by various replicons, the great majority of genes encoding CTX-M-14 and CTX-M-15 ESBLs were carried by IncF replicons, as were TEM-1 and IRT β-lactamases.
Conclusions: Resistance genes borne by the narrow host-range IncF replicon spread readily as this replicon is well adapted to E. coli. This is observed for blaTEM-1 and blaCTX-M-15 and, to a lesser extent, for blaCTX-M-14. Transposition immunity seems to play an important role in the diffusion process.
Key Words: PCR-based replicon typing , ESBLs , E. coli , transposition immunity
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Until the late 1990s, TEM and SHV enzymes were the most common extended-spectrum β-lactamases (ESBLs), frequently associated with Klebsiella pneumoniae.1 In the late 1990s, the prevalence of both TEM and SHV decreased, whereas that of CTX-M increased, especially associated with Escherichia coli species. Within a few years, CTX-M ESBL-producing E. coli had spread across the world, involved in both nosocomial outbreaks and community-acquired infections.1
To investigate the mechanisms involved in the emergence and spread of these plasmid-borne ESBLs, we studied a collection of 125 ESBL-producing E. coli strains2 and in particular: (i) the replicons carrying the ESBLs; and (ii) the total plasmid content of the strains, in relation to the type of ESBL. The ESBL replicons were then compared with the replicons carrying TEM-1, a TEM ESBL ancestor, and inhibitor-resistant TEM (IRT) β-lactamases, another type of TEM-derived enzyme that emerged in the 1990s after clavulanic acid started to be used.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Bacterial strains
One hundred and twenty-five strains of ESBL-producing E. coli were studied.2 They were recovered between 1997 and 2002 in different areas of France and were not epidemiologically related. The ESBL genes in these strains had been characterized previously (Table 1).2 E. coli J53-2 rifr (Stratagene, La Jolla, CA, USA) and E. coli DH10BTM (Invitrogen, Cergy Pontoise, France) were used for conjugation and transformation experiments, respectively.
|
In addition, two other collections of strains were studied for comparison: (i) 44 non-repetitive (one strain, one patient) TEM-1-producing clinical E. coli strains isolated between January and March 2003 in Tenon Hospital (Paris, France) and with different profiles by Rep-PCR; and (ii) 39 non-repetitive IRT β-lactamase-producing clinical E. coli strains (14 IRT-2, 7 IRT-4, 4 IRT-5, 7 IRT-6, 3 IRT-7, 3 IRT-8 and 1 IRT-10) isolated between 1995 and 2002 at Saint-Louis and Tenon Hospitals (Paris, France). The presence of the blaTEM gene in these TEM-1- and IRT-producing strains was characterized as described previously.3
ESBL resistance transfer assays
The plasmid conferring third-generation cephalosporin resistance in each of the 125 ESBL-producing strains was transferred by mating with E. coli J53-2 rifr. For donor strains producing TEM- or SHV-type ESBLs, transconjugants were selected on Drigalski agar plates (Bio-Rad) containing rifampicin (250 mg/L) and ceftazidime (2 mg/L), and for CTX-M-producing donors, they were selected on Drigalski agar plates containing rifampicin (250 mg/L) and cefotaxime (2.5 mg/L). When resistance plasmid transfer failed in the mating experiments (36 cases) or when plasmid co-transfer occurred (11 cases), transformation was used: plasmid DNA was extracted and then transferred into E. coli DH10BTM cells by electroporation (Invitrogen). Transformants were selected on Drigalski agar plates supplemented with a third-generation cephalosporin, as described earlier.
Detection of TEM-1 in ESBL-producing strains
Isoelectric focusing was used to detect TEM-1 in all parental strains and in TEM ESBL recipients. PCR and direct sequencing using OT3 and OT4 primers were used to detect blaTEM-1 in CTX-M and SHV ESBL-producing parental and recipient strains.3
Plasmid replicon type determination
Plasmid replicons were determined using the PCR-based replicon typing scheme described by Carattoli et al.4 and primers, CA1 and OR1, for the FII replicon.5 The latter primers recognize FII replicons sharing high-level homology with the R100 reference plasmid, whereas the repF simplex PCR described by Carattoli et al.4 could detect more divergent FII replicons, and all positive results were collected in the same repFII replicon type.
Plasmid replicons were determined for the ESBL-producing parental strains and for transconjugants and transformants obtained after transfer of resistance as well as for E. coli clinical isolates producing TEM-1 and IRT enzymes.
All comparisons were determined using Pearson's 
2 or Fisher's exact test when appropriate, and a P value < 0.05 was considered significant. Statistical analysis was carried out using STATA v 9.2 (StataCorp., College Station, TX, USA).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
PCR-based replicon typing in parental ESBL-producing strains
The replicons could not be determined in 7 (5.6%) of the 125 parental strains tested. In total, 291 replicons were detected (mean 2.33, range 0–6) [Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].
F replicons (FIA, FIB, FIC and FII) were the most frequently detected replicon types in our collection (60.5%). These results are in accordance with those of other authors.6,7 IncF replicons are widely distributed among E. coli strains and seem to be well adapted to this species.8 Curiously, the A/C replicon was detected in 48 of the parental strains (38.4%), although this replicon has been considered to be rare in Enterobacteriaceae and particularly in E. coli isolated from humans.6,7
PCR-based replicon typing in ESBL-recipient strains
The ESBL plasmid replicons could not be characterized for 25 (20%) of the 125 ESBL plasmids in the transconjugants/transformants strains tested; many of these were SHV ESBL producers (9/21) (Table 1). The proportion on non-characterized replicons of SHV ESBL producers (43%) was significantly greater than that observed for TEM ESBLs (P = 0.009) and CTX-M ESBLs (P = 0.02) and suggests that these genes are carried by plasmids that this PCR-based method is unable to type (Table 1). Although SHV-type ESBLs, including SHV-2, SHV-5 and SHV-12, are disseminated worldwide, little is known about the plasmids encoding these ESBLs.
A/C replicons were associated with the three ESBL types studied (TEM, SHV and CTX-M), but were significantly more prevalent in TEM ESBL plasmids (36/54) (ESBL type versus TEM: SHV, P < 0.001; CTX-M, P < 0.001). As most of the IncA/C replicons were associated with TEM-24-producing strains, the apparent over-representation of this type of replicons in our collection could be a bias (Table 1). Similar to A/C replicons, I1 replicons were associated with several β-lactamases in our and other studies.9,10 Particularly, all TEM-52 genes for which plasmid replicon type could be determined (7/10) were carried on IncI1 replicons (Table 1).
Conversely, IncF replicons did not carry all types of ESBLs. Indeed, among the 54 TEM ESBL replicons that we studied, none belonged to the IncF group. Although CTX-M enzymes can be carried by various replicons, most (36%) were carried by IncF replicons (FIA, FIB and FII). Diverse replicons have been implicated in CTX-M emergence. Most of the CTX-M-9 genes in Great Britain and Spain are carried on HI2 replicons.10,11 However, in our collection, most were on IncF replicons. The number of CTX-M-9-producing strains in our collection was too small (Table 1) to draw conclusions. CTX-M-1 and CTX-M-3 are early enzymes of the cluster, whereas CTX-M-15 appeared more recently, evolving from CTX-M-3 by an Asp-240-Gly substitution, which increases its catalytic efficiency against ceftazidime. The CTX-M-1 and CTX-M-3 genes in our collection were carried on various replicons (IncI1, and IncN, or IncL/M, respectively) but not on IncF, whereas most of the CTX-M-15 enzymes are encoded on IncF replicons (Table 1). To the best of our knowledge, no CTX-M-1 or CTX-M-3 has been described on an IncF replicon.12
PCR-based replicon typing in E. coli producing TEM-1 and IRT β-lactamases
We performed PCR replicon typing with the 44 TEM-1-producing clinical isolates of E. coli: none of them carried A/C replicons, whereas 31 (70%) carried F replicons; 87% of the typed replicons in TEM-1 producers were F replicons. Replicon type was also determined for 39 IRT-producing clinical isolates of E. coli. As observed for strains producing TEM-1 enzyme, no A/C replicons were detected, whereas 35 strains (90%) contained F replicons, and 96% of the typed replicons found in IRT producers were F replicons (Table 2). There were significant differences between IRT producers versus TEM-1 or TEM ESBL producers for FIB replicons (P < 0.0001). For FII replicons, there were significant differences between TEM ESBL producers versus IRT producers (P < 0.0001) and TEM ESBL producers versus TEM-1 producers (P < 0.05) (Table 2).
|
Implications for understanding of ESBL emergence and dissemination
blaTEM-1 genes, which have successfully diffused in E. coli all over the world during the last 40 years, are mainly carried by F replicons. These narrow host-range replicons are well adapted to E. coli species and can easily conjugate. We show here that IRT β-lactamases are carried by the same replicons as TEM-1. As β-lactamase inhibitors, and in particular clavulanic acid, are extensively used (
96%) in human medicine in France (and probably in other industrialized countries), we can hypothesize that IRT β-lactamases have evolved in vivo in the same plasmid platforms carrying blaTEM-1 genes. In contrast, the observation that TEM ESBLs do not share the same replicons as TEM-1 (A/C versus F) suggests that TEM ESBLs may well have appeared outside human E. coli. There are no data available on the replicon type of plasmids encoding TEM ESBLs in E. coli outside humans. However, some IncA/C and IncL/M replicons have been associated with TEM ESBLs in K. pneumoniae, and TEM-52-carrying IncI1 replicons have been described in Salmonella.13,14 Furthermore, as TEM genes are carried by Tn3-type transposons, known to confer transposition immunity (a plasmid containing a copy of Tn3 is resistant to further insertions of Tn3), it is plausible that TEM ESBL transposition from broad host-range A/C or I1 replicons to F replicons already carrying TEM-1 is not possible.15
To test this hypothesis, we investigated the presence of TEM-1 in our ESBL collection. Sixty-one parental strains (49%) produced a β-lactamase with a pI of 5.4, compatible with the presence of the TEM-1 enzyme. The prevalence was not different between the three groups: 41% for TEM ESBL carriers, 52% for SHV ESBL producers and 56% for CTX-M ESBL carriers [Table S2, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. TEM-1 β-lactamase was detected in 12 (80%) of the CTX-M-15-producing parental strains, in 6 (43%) of the CTX-M-14 producers and in 10 (48%) of the other CTX-M producers. The high prevalence of TEM-1 in CTX-M-15 producers has been reported previously.16–19
We then determined whether blaTEM-1 was carried on the same replicon as the ESBL gene. In TEM ESBL-producing recipient strains, we did not detect any band of β-lactamase activity compatible with TEM-1 (pI 5.4). Conversely, 8 of the 12 CTX-M-15-recipient strains obtained from parental strains producing both CTX-M-15 and TEM-1 also produced TEM-1 β-lactamase, indicating linkage of the blaTEM-1 and blaCTX-M-15 genes on the same replicon, as described previously.18,19 These results suggest that two copies of the blaTEM genes cannot be located in the same replicon.
Kluyvera spp. are the ancestral host of CTX-M enzymes, so we know that these resistance genes came from the environment.2 Some of these genes, for example, blaCTX-M-1 and blaCTX-M-3, were mobilized by ISEcp1 probably on non-IncF replicons (IncL/M, IncN and IncI1).2 Subsequent transposition from these replicons to F replicons well adapted to E. coli is not hampered by Tn3 transposition immunity.
In summary, it appears that resistance genes borne by the narrow host-range IncF replicons can spread readily in E. coli, as observed for blaTEM-1 and blaCTX-M-15. Also, transposition immunity plays an important role in the dissemination process, as shown for TEM ESBL.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
G. M. was supported by a grant from the ‘Fondation pour la Recherche Médicale’. This work was partially funded by ‘Contrat d'Initiation à la Recherche Clinique’ CRC 05 103 (Assistance Publique, Hôpitaux de Paris), the ‘Fondation pour la Recherche Médicale’ and the Faculté de Médecine Pierre et Marie Curie, Université Paris 6.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Tables S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Acknowledgements |
|---|
We are grateful to A. Carattoli for providing the plasmid incompatibility group controls.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
|---|
1 . Cantón R, Coque TM. The CTX-M β-lactamase pandemic. Curr Opin Microbiol (2006) 9:466–75.[CrossRef][Web of Science][Medline]
2 . Branger C, Zamfir O, Geoffroy S, et al. Genetic background of Escherichia coli and extended-spectrum β-lactamase type. Emerg Infect Dis (2005) 11:54–61.[Web of Science][Medline]
3
.
Eckert C, Gautier V, Arlet G. DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother (2006) 57:14–23.
4 . Carattoli A, Bertini A, Villa L, et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods (2005) 63:219–28.[CrossRef][Web of Science][Medline]
5
.
Osborn AM, da Silva Tatley FM, Steyn LM, et al. Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons. Microbiology (2000) 146:2267–75.
6 . Sherley M, Gordon DM, Collignon PJ. Species differences in plasmid carriage in the Enterobacteriaceae. Plasmid (2003) 49:79–85.[Medline]
7
.
Johnson TJ, Wannemuehler YM, Johnson SJ, et al. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates. Appl Environ Microbiol (2007) 73:1976–83.
8 . Boyd EF, Hill CW, Rich SM, et al. Mosaic structure of plasmids from natural populations of Escherichia coli. Genetics (1996) 143:1091–100.[Abstract]
9 . Carattoli A, Miriagou V, Bertini A, et al. Replicon typing of plasmids encoding resistance to newer β-lactams. Emerg Infect Dis (2006) 12:1145–8.[Web of Science][Medline]
10
.
Hopkins KL, Liebana E, Villa L, et al. Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates. Antimicrob Agents Chemother (2006) 50:3203–6.
11
.
Novais A, Canton R, Valverde A, et al. Dissemination and persistence of blaCTX-M-9 are linked to class 1 integrons containing CR1 associated with defective transposon derivatives from Tn402 located in early antibiotic resistance plasmids of IncHI2, IncP1-
, and IncFI groups. Antimicrob Agents Chemother (2006) 50:2741–50.
12
.
Novais A, Canton R, Moreira R, et al. Emergence and dissemination of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in Spain are associated with IncFII (CTX-M-15) and broad-host-range (CTX-M-1, -3, and -32) plasmids. Antimicrob Agents Chemother (2007) 51:796–9.
13
.
Sirot D, Chanal C, Labia R, et al. Comparative study of five plasmid-mediated ceftazidimases isolated in Klebsiella pneumoniae. J Antimicrob Chemother (1989) 24:509–21.
14
.
Cloeckaert A, Praud K, Doublet B, et al. Dissemination of an extended-spectrum-β-lactamase blaTEM-52 gene-carrying IncI1 plasmid in various Salmonella enterica serovars isolated from poultry and humans in Belgium and France between 2001 and 2005. Antimicrob Agents Chemother (2007) 51:1872–5.
15
.
Kans JA, Casadaban MJ. Nucleotide sequences required for Tn3 transposition immunity. J Bacteriol (1989) 171:1904–14.
16
.
Carattoli A, García-Fernández 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.
17
.
Karisik E, Ellington MJ, Pike R, et al. Molecular characterization of plasmids encoding CTX-M-15 β-lactamases from Escherichia coli strains in the United Kingdom. J Antimicrob Chemother (2006) 58:665–8.
18
.
Boyd DA, Tyler S, Christianson S, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum β-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother (2004) 48:3758–64.
19
.
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, Tunis, and Bangui. Antimicrob Agents Chemother (2006) 50:2433–8.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Valverde, R. Canton, M. P. Garcillan-Barcia, A. Novais, J. C. Galan, A. Alvarado, F. de la Cruz, F. Baquero, and T. M. Coque Spread of blaCTX-M-14 Is Driven Mainly by IncK Plasmids Disseminated among Escherichia coli Phylogroups A, B1, and D in Spain Antimicrob. Agents Chemother., December 1, 2009; 53(12): 5204 - 5212. [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. Hrabak, J. Empel, T. Bergerova, K. Fajfrlik, P. Urbaskova, I. Kern-Zdanowicz, W. Hryniewicz, and M. Gniadkowski International Clones of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum {beta}-Lactamases in a Czech Hospital J. Clin. Microbiol., October 1, 2009; 47(10): 3353 - 3357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ruppe, P.-L. Woerther, A. Diop, A.-M. Sene, A. Da Costa, G. Arlet, A. Andremont, and B. Rouveix Carriage of CTX-M-15-Producing Escherichia coli Isolates among Children Living in a Remote Village in Senegal Antimicrob. Agents Chemother., July 1, 2009; 53(7): 3135 - 3137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Carattoli Resistance Plasmid Families in Enterobacteriaceae Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2227 - 2238. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||