JAC Advance Access published online on November 6, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn453
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Characterization of plasmids encoding blaESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae
1 Servicio de Microbiología, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 2 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain 3 Servicio de Microbiología, Hospital Son Dureta, Palma de Mallorca, Spain 4 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain 5 CIBER en Epidemiología y Salud Pública (CIBERESP), Spain 6 Laboratorio de Antibióticos, Servicio de Bacteriología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 7 Unidad de Resistencia a Antibióticos y Virulencia Bacteriana Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
* Correspondence address. Servicio de Microbiología, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Tel: +34-932919071; Fax: +34-932919070; E-mail: fnavarror{at}santpau.cat
Received 22 August 2008; returned 15 September 2008; revised 3 October 2008; accepted 7 October 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Objectives: The aim of the study was to characterize plasmids that harbour blaESBL genes and their genetic environment in Escherichia coli and Klebsiella pneumoniae clones circulating in Spain.
Methods: The incompatibility group of plasmids within 58 strains harbouring blaCTX-M (n = 45) and blaSHV (n = 15) genes was determined by rep-typing-PCR and hybridization. The blaESBL genetic environment was determined by PCR and sequencing.
Results: The blaCTX-M-9 genes (n = 14) were linked to In60 located in IncI1 (50%) or IncHI2 plasmids (28%). All blaCTX-M-14 genes (n = 13) were flanked by ISEcp1 and IS903 and 12 were associated with IncK plasmids. One of two blaCTX-M-10 genes was present in an IncK plasmid, but both genes were linked to a phage-related element. Five of seven blaCTX-M-1 (71%), all three blaCTX-M-32 and one of two blaCTX-M-3 genes were linked to IncN plasmids. The other blaCTX-M-3 gene was linked to IncA/C and the remaining two blaCTX-M-1 genes to IncFII plasmids. Three blaCTX-M-15 genes were associated with IncF (repFIA) and one with IncFII plasmids. All these genes from blaCTX-M group-1 showed the ISEcp1 upstream truncated by different insertion sequences. Forty-three percent of blaSHV-12 genes (n = 14) were located in IncI1 plasmids, all flanked by the IS26 and DEOR region. The only detected blaSHV-5 gene was located in an IncFII plasmid and flanked by recF and DEOR regions.
Conclusions: A diversity of the plasmid incompatibility groups that harbour blaESBL genes was observed, except for the blaCTX-M-14 gene. Moreover, a high variability was confirmed in the genetic environment of these genes as a result of insertion and deletion events.
Key Words: ESBL-producing , incompatibility group , antimicrobial resistance surveillance , mechanisms of resistance
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Distribution and prevalence of extended-spectrum β-lactamase (ESBL) have changed dramatically in recent years. This process could have been influenced by the massive use of certain antibiotics; however, others factors must also be taken into account. The association of blaESBL genes with genetic elements, which potentially facilitate the capture and expression of blaCTX-M genes as integrons or insertion sequences, including ISEcp1, ISCR1 or IS26, might also have played a role.1 It has been well-documented in several studies that the spread, in general, is not only due to the expansion of a clonal strain, but also to plasmid dissemination.2–5
In our previous study,6 a great clonal diversity among the Escherichia coli strains collected in 2004 was observed, the predominant ESBL being CTX-M-14 followed by CTX-M-9 and SHV-12. Regarding Klebsiella pneumoniae strains, a lower clonal diversity was observed but with higher diversity of the enzymes including CTX-M-type (CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15), SHV-type (SHV-12 and SHV-5) and TEM-4. Three clusters included 53.1% of strains. The high clonal diversity of ESBL producers could be due to the presence of blaESBL genes in plasmids and further dissemination in different E. coli and K. pneumoniae clones. The identification of the plasmid incompatibility group and the characterization of the surrounding regions of blaESBL genes enable the tracking of the genes' mobility.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Isolates
From a collection of 92 E. coli and 32 K. pneumoniae isolates recovered from 11 Spanish hospitals over the first 3 months of 2004 during a nationwide survey within the Spanish Network in Infectious Pathology Project,6 we selected 58 representative strains (see below) carrying different ESBL of the families CTX-M (n = 45: 14 CTX-M-9, 13 CTX-M-14, 7 CTX-M-1, 4 CTX-M-15, 3 CTX-M-32, 2 CTX-M-3 and 2 CTX-M-10) and SHV (n = 15: 14 SHV-12 and 1 SHV-5). Two strains had both CTX-M-9 and SHV-12 enzymes. Only one isolate per clone was selected. Clonality was studied in the previously mentioned study.6
Genetic environment characterization
The genetic context of blaESBL was investigated by searching for the presence and linkage with sequences previously reported to be associated with the CTX-M group genes, such as ISEcp1, IS903, IS26, ISCR1 or orf1005. PCR and sequencing using previously described primers were employed to investigate these surrounding regions.1,2,7 Additionally, primers designed in accordance with accessible DNA sequences in the GenBank (EF370423 [GenBank] , AY532647 [GenBank] , AJ245670 [GenBank] ) were used to ascertain the presence of genes linked to the SHV group (Table 1). For strains showing negative PCR results, the corresponding blaESBL genes were cloned in order to investigate their surrounding regions. For this purpose, plasmid DNA of each strain [obtained with the QIAfilter Plasmid Midi Kit (Qiagen, Hilden, Germany)] was digested with BamHI, EcoRI or HindIII and further ligated to pBGS18 digested with the same enzyme. Obtained products were then transformed into XL1 Blue E. coli strain made competent by CaCl2. Transformants were selected in 1 mg/L cefotaxime–30 mg/L kanamycin LB agar plates. After checking plasmid DNA from the transformants for the presence of the corresponding ESBL gene in the cloned DNA fragments, the surrounding regions were sequenced using reverse blaESBL primers (Table 1).
|
Characterization of plasmids carrying ESBL
The present study included 24 wild-type strains (15 E. coli and 9 K. pneumoniae) and 34 transconjugants (26 E. coli and 8 K. pneumoniae). The latter were obtained using E. coli HB101 kanamycin-azide-resistant strain as recipient.2
In all 58 strains, the incompatibility group of plasmids carrying blaESBL genes was determined by a PCR-based method as described previously.8 Plasmid profiles of the transconjugants and wild-type strains were determined by PFGE of S1 nuclease digested genomic DNA. PFGE was performed on a CHEF DRIII apparatus (Bio-Rad Laboratories, Munich, Germany) using a run time of 24 h, 14°C, 120° of angle and a 6 V/cm of voltage, with initial and final switch times of 5–25 s for 6 h and 30–45 s for 18 h. Lambda ladder (New England Biolabs, Ipswich, USA) was used as a marker. Gels were transferred by Southern blot, and the membranes obtained were hybridized with probes specific for blaCTX-M, blaSHV and rep sequences. We used the amplified replicons as probes. Probe labelling, hybridization and detection were performed with the ECL kit (GE Healthcare Amersham ECLTM, Buckinghamshire, UK) as described previously.2
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
As mentioned earlier, the 58 studied strains were selected from a collection of 92 E. coli and 32 K. pneumoniae isolates recovered from 11 Spanish hospitals over a trimester of 2004.6 This study showed a higher diversity of ESBL-types in both E. coli and K. pneumoniae than that observed in a previous epidemiological study performed in 2000 in Spain,6 in which the most predominant ESBL were CTX-M-9 (27.3%), SHV-12 (23.9%) and CTX-M-14 (20.5%) for E. coli, and TEM-3 (16.7%) and TEM-4 (25%) for K. pneumoniae. The corresponding enzymes in the 2004 study6 were CTX-M-14 (45.7%), SHV-12 (21.7%) and CTX-M-9 (20.6%) for E. coli and CTX-M-1 (31.3%), CTX-M-9 (21.9%) and SHV-12 (28.1%) for K. pneumoniae. Moreover, there were no epidemiological relationships or evidence of clonality among the ESBL-producing E. coli isolates, although clonal relationships were observed among some K. pneumoniae isolates.6 This suggests, as previously stated by other authors,1 that plasmids and/or mobile elements could be involved in the spread of blaESBL genes. To ascertain this possibility, characterization of plasmids carrying these genes and their corresponding genetic environment were performed from 34 transconjugants and 25 wild-type isolates. Table 2 and Figure 1 summarize the results obtained from all these strains.
|
|
The 14 blaCTX-M-9 genes detected in different clones were located in plasmids of the incompatibility groups IncI1 (n = 7; 50%), IncHI2 (n = 4; 28.5%), IncP (n = 3; 21.4%), IncF (repFIB) (n = 2; 14.3%) and IncK (n = 1; 7.1%), highlighting the presence of replicons corresponding to IncI1 and IncP plasmids in the same plasmid in three cases. The only four IncHI2 plasmids containing blaCTX-M-9 were isolated from three hospitals from Barcelona, and no other IncHI2 plasmids associated with the other blaESBL genes were detected. The plasmids' association between IncI1 or IncHI2 plasmids and blaCTX-M-9 observed in this study has already been described in our country and highlights the persistence of this association.4,9,10 The analysis of the genetic environment revealed that blaCTX-M-9 genes were associated with In60 related integrons, but a great diversity within this element was observed in agreement with previous Spanish studies.2 Variations were mainly related to deletions within the upstream region (ISCR1). In our study, one strain did not contain the ISCR1 and orf1005 genes upstream and downstream of blaCTX-M-9, respectively; five strains did not contain the orf1005 gene downstream and eight strains contained both genes (Figure 1).
It is noteworthy that, with the exception of one blaCTX-M-14 gene in which it was not possible to determine the incompatibility group, all were associated with IncK plasmids. This close association between blaCTX-M-14 and IncK plasmid has also been previously documented in different studies from single institutions.9,10 Moreover, the surrounding regions of blaCTX-M-14 were also highly conserved. ISEcp1 was found upstream and IS903 downstream of the gene, as has been documented previously (Figure 1).1,3
The seven blaCTX-M-1 genes were located in plasmids of IncN (n = 5; 71.4%) and IncFII (n = 2; 28.6%). All three blaCTX-M-32 genes were also found in IncN plasmids. The 3' region of the ISEcp1 insertion sequence truncated by IS26 was found 80 bp upstream in all seven blaCTX-M-1 genes. IS26 was inserted in these seven strains in the same position (at nucleotide 214 from the end of ISEcp1), but a 74 bp duplication of the beginning of the IS26 transposase was observed in five of them (Figure 1). Moreover, in these five strains (in contrast to the other two strains), IS26 was preceded by two genes (DNA invertase invA and orf10) showing 100% identity to those detected in a Comamonas acidovorans strain DNA sequence (AB063332 [GenBank] ). Similar to blaCTX-M-1 genes, all three blaCTX-M-32 environments showed ISEcp1 at 80 bp upstream of the gene (with identical nucleotide sequence), but truncated by IS5 instead of IS26, and in a different position (at nucleotide 152 from the end of ISEcp1) (Figure 1). The location of both blaCTX-M-1 and blaCTX-M-32 genes on related broad-host-range IncN plasmids has also been described in Madrid hospitals.3 This emphasizes the relevance of the evolution of blaESBL genes within specific genetic environments, as has also been suggested for blaCTX-M-14 and blaCTX-M-9.10
Among blaCTX-M-3 (n = 2), one was located in an IncN plasmid, 48 bp downstream of ISEcp1 truncated by IS26, similar to two of the blaCTX-M-1 environment. The other one was located in the IncA/C plasmid, 45 bp downstream of ISEcp1 (Figure 1). Plasmids from the IncA/C group seem to be widely spread as they have been detected in CTX-M-2, TEM-24 or CMY-producing isolates from different European and/or American countries.3
Three blaCTX-M-15 genes were located in IncF (repFIA) and one in IncFII plasmids. Different recent studies have pointed out that pandemic dissemination of CTX-M-15-producing Enterobacteriaceae is linked to epidemic E. coli B2 strain and/or epidemic IncFII plasmids, although association with IncFI plasmids has recently been described.3,5,9,11 It is noteworthy that the four E. coli carrying blaCTX-M-15 genes were not clonally related.6 Regarding the blaCTX-M-15 environment, the right boundary of ISEcp1 was located 48 bp upstream of the gene in all cases, as already described in Spanish, French, Indian and Turkish isolates.1,3,5 One strain had ISEcp1 truncated by an IS26 at nucleotide 24 from the end of ISEcp1 (Figure 1). Thus, ISEcp1 was always found upstream of blaCTX-M coding for CTX-M-1-like enzymes (blaCTX-M-1, blaCTX-M-3, blaCTX-M-15, blaCTX-M-32) and is strongly implicated in the mobilization of this antibiotic resistance gene.1
One of the two blaCTX-M-10 genes was located in the IncK plasmid, but it was not possible to determine the incompatibility group of the other one. As described previously,7 a gene encoding a phage-related DNA invertase was detected in the two strains upstream of the bla gene (Figure 1), suggesting the mobilization of blaCTX-M-10 to a transferable plasmid may have been mediated by transduction.
Twelve of the 14 blaSHV-12 genes were located in IncI1 (n = 6, 42.9%), IncK (n = 5, 35.7%) and IncFII plasmid (n = 1, 7.1%). The two remaining strains simultaneously carried the blaSHV-12 and the blaCTX-M-9 gene. In one of them, both genes were located in the same plasmid, the IncHI2 plasmid (n = 1, 7.1%), but it was not possible to determine the incompatibility group of the other one. These results are in agreement with another study that correlated blaSHV-12 with a diversity of IncFII, IncI1 and IncA/C plasmids.8 IS26 was found 73 bp upstream of the blaSHV-12 gene in all cases. In all but two SHV-12-producing strains, the putative DEOR transcriptional regulator (GenBank EF370423 [GenBank] ) was identified 21 bp downstream of the blaSHV-12 gene.
The blaSHV-5 gene was located in IncFII plasmid and its environment was surrounded by a gene encoding a putative RecF protein (GenBank AY532647 [GenBank] ) upstream and the gene coding the putative DEOR transcriptional regulator downstream (Figure 1).
In conclusion, our study highlights circulation of different plasmids containing a diversity of blaESBL genes associated with different genetic environments in Spain. The diversity of genetic contexts found for particular bla genes located in specific plasmids suggests the evolution of these plasmids by different recombinatorial events.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
This study was partially supported by grants FIS PI04339, PI040162, PI040837 and PI042012, by AGAUR 2006FI00426 and also by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III - FEDER, Spanish Network for the Research in Infectious Diseases REIPI C03/14 and Spanish Network for Research in Infectious Diseases REIPI RD06/0008.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We are grateful to the participants in this project for their help during the collection of the isolates: L. Matas and M. Giménez (Hospital Germans Trias i Pujol, Badalona), I. Sanfeliu (Corporació Hospitalària Parc Taulí, Sabadell), G. Prats (Hospital Vall d’Hebron, Barcelona), M. Ruíz and J. Vila (Hospital Clínic, Barcelona), A. Domínguez (Hospital de Bellvitge, Hospitalet), A. Guerrero and J. Colomina (Hospital de La Ribera, Alzira), A. Pascual (Hospital Virgen de la Macarena, Sevilla) and J. Aznar (Hospital Virgen del Rocío, Sevilla).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
1 . Lartigue M, Poirel L, Nordmann P. Diversity of genetic environment of blaCTX-M genes. FEMS Microbiol Lett (2004) 234:201–7.[CrossRef][Web of Science][Medline]
2
.
García A, Navarro F, Miró E, et al. Characterisation of the highly variable region surrounding the blaCTX-M-9 gene in non-related Escherichia coli from Barcelona. J Antimicrob Chemother (2005) 56:819–26.
3
.
Novais A, Cantón 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.
4 . García A, Navarro F, Miró E, et al. Acquisition and diffusion of blaCTX-M-9 gene by R478-IncHI2 derivative plasmids. FEMS Microbiol Lett (2007) 271:71–7.[CrossRef][Web of Science][Medline]
5 . Coque TM, Novais A, Carattoli A, et al. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum β-lactamase CTX-M-15. Emerg Infect Dis (2008) 14:195–200.[Web of Science][Medline]
6 . Diestra K, Coque TM, Miró E, et al. Caracterización y epidemiología molecular de BLEE en Escherichia coli y Klebsiella pneumoniae en 11 hospitales españoles. Enferm Infecc Microbiol Clin (2008) 26:404–10.[CrossRef][Web of Science][Medline]
7
.
Oliver A, Coque TM, Alonso D, et al. CTX-M-10 linked to a phage-related element is widely disseminated among Enterobacteriaceae in a Spanish hospital. Antimicrob Agents Chemother (2005) 49:1567–71.
8 . 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]
9
.
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.
10 . Navarro F, Mesa RJ, Miró E, et al. Evidence for convergent evolution of CTX-M-14 ESBL in Escherichia coli and its prevalence. FEMS Microbiol Lett (2007) 273:120–3.[CrossRef][Web of Science][Medline]
11
.
Gonullu N, Aktas Z, Kayacan CB, et al. Dissemination of CTX-M-15 β-lactamase genes carried on Inc FI and FII plasmids among clinical isolates of Escherichia coli in a University Hospital in Istanbul, Turkey. J Clin Microbiol (2008) 46:1110–2.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Yong, M. A. Toleman, C. G. Giske, H. S. Cho, K. Sundman, K. Lee, and T. R. Walsh Characterization of a New Metallo-{beta}-Lactamase Gene, blaNDM-1, and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from India Antimicrob. Agents Chemother., December 1, 2009; 53(12): 5046 - 5054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
L. F. Mataseje, N. Neumann, B. Crago, P. Baudry, G. G. Zhanel, M. Louie, M. R. Mulvey, and and the ARO Water Study Group Characterization of Cefoxitin-Resistant Escherichia coli Isolates from Recreational Beaches and Private Drinking Water in Canada between 2004 and 2006 Antimicrob. Agents Chemother., July 1, 2009; 53(7): 3126 - 3130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Carattoli Resistance Plasmid Families in Enterobacteriaceae Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2227 - 2238. [Full Text] [PDF]
|
|
|
|
|
|
J. Rodriguez-Bano, J. Alcala, J. M. Cisneros, F. Grill, A. Oliver, J. P. Horcajada, T. Tortola, B. Mirelis, G. Navarro, M. Cuenca, et al. Escherichia coli producing SHV-type extended-spectrum {beta}-lactamase is a significant cause of community-acquired infection J. Antimicrob. Chemother., April 1, 2009; 63(4): 781 - 784. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||