JAC Advance Access published online on March 26, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn131
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum β-lactamases in Escherichia coli and Salmonella of human and animal origin
1 Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy 2 Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy
* Corresponding author. Tel: +39-06-49903128; Fax: +39-06-49387112; E-mail: alecara{at}iss.it
Received 17 January 2008; returned 28 February 2008; revised 11 February 2008; accepted 29 February 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Objectives: Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of β-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment.
Methods: pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for β-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST.
Results: Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried blaCMY-2, blaCTX-M-15, blaCTX-M-1, blaCTX-M-14, blaTEM-52, blaSHV-12 or blaTEM-1 β-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST.
Conclusions: The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among β-lactamase producers isolated from animals and humans.
Key Words: pMLST , incompatibility group , CTX-M , CMY , SHV
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Plasmids belonging to incompatibility group I1 (IncI1) carrying extended-spectrum and AmpC β-lactamase genes have recently been described in Escherichia coli and Salmonella.1,2 Three typical IncI1 plasmids were fully sequenced: R64 and colIb-P9, lacking β-lactamase genes, and pNF1358 carrying the blaCMY2 gene. These plasmids are characterized by the presence of a cluster encoding the type IV pili, contributing to adhesion and invasion of Shiga-toxigenic E. coli.3 These peculiar pili are a virulence factor and the association of virulence and resistance determinants may favour the positive selection of plasmids belonging to the IncI1 family.4 A study performed in the USA on a large collection of avian and human E. coli demonstrated that IncI1 plasmids were more frequent in pathogenic than in commensal strains.4 The observation that IncI1 plasmids have been recently associated with highly widespread β-lactamase genes in E. coli and Salmonella from food animals is of concern for the potential spread of resistance determinants through the food chain.
The aim of this work was to analyse and characterize IncI1 plasmids identified in β-lactamase E. coli and Salmonella producers from animal and human sources in Europe and the USA. We set up a new plasmid multilocus sequence typing (pMLST) method to rapidly categorize plasmids belonging to the IncI1 family with different sequence types (STs).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Plasmids
Sixteen IncI1 plasmids were analysed for β-lactamases and plasmid scaffold. The blaTEM, blaSHV, blaCTX-M, blaCMY and blaOXA genes were searched for by PCR,5 and amplicons were sequenced by fluorescent dye-labelled dideoxynucleotides using an ABI 3730 instrument (Applied Biosystems). The 16 plasmids were assigned to the IncI1 group by the PCR-based replicon typing (PBRT) method.6 Twelve plasmids had been previously characterized: eight of them were kindly provided by L. Poirel (INSERM U914, Bicetre, France),7 A. Cloeckaert (INRA, Nouzilly, France)1 and K. Hopkins (HPA, Colindale, London, UK),2 and four were from the collection of the Istituto Superiore di Sanità (18196T, 1358T, R144 and S82/10).6,8–10 Four transconjugant strains (398T, 3115T, 3960T and 2392T) were obtained using E. coli K12 as a recipient strain from β-lactamase E. coli producers of animal origin isolated at the Istituto Zooprofilattico delle Venezie, Italy, in 2005–2006. Plasmids 398T and 3115T were positive for the blaCMY-2 gene, and 3960T and 2392T were positive for the blaCTX-M-1 gene and the blaSHV-12 and blaTEM-1 genes, respectively (Table 1).
|
Plasmid DNA was purified by the QIAGEN Plasmid Midi Kit (Qiagen Inc., Milano, Italy) and analysed by restriction fragment length polymorphism (RFLP) by PstI digestion and Southern blot hybridization using the amplicons obtained from repI1 and blaCMY-2 and blaCTX-M-1-group genes, respectively, as probes. Plasmids showing no more than two bands of difference were assigned to the same RFLP type (Table 1).
Plasmid multilocus sequence typing
Primers for pMLST are listed in Table 2. We selected 254 bp of pilL, 254 bp of sogS and 343 bp of ardA gene coding sequences. Moreover, a 104 bp sequence of repI1 and a 812 bp sequence including the 3' end of the trbA gene, the intergenic region and the 5' end of the pndC gene were also included as pMLST targets.
|
PCRs were performed as follows: 1 cycle at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and elongation at 72°C for 1 min. The amplification was concluded by 1 cycle at 72°C for 5 min. The amplicons were purified using the Wizard PCR Preps DNA purification system (Promega, Madison, WI, USA) and fully sequenced.
The allele variants were assigned to the following EMBL accession numbers: repI1-1 (EU370458 [GenBank] ), repI1-2 (EU370459 [GenBank] ), repI1-3 (EU370460 [GenBank] ), ardA1 (EU370452 [GenBank] ), ardA2 (EU370453 [GenBank] ), ardA3 (EU370454 [GenBank] ), trbA-pndC1 (EU370465 [GenBank] ), trbA-pndC2 (EU370466 [GenBank] ), trbA-pndC3 (EU370467 [GenBank] ), trbA-pndC4 (EU370468 [GenBank] ), trbA-pndC5 (EU370469 [GenBank] ), trbA-pndC6 (EU370452 [GenBank] ), sogS1 (EU370461 [GenBank] ), sogS2 (EU370462 [GenBank] ), sogS3 (EU370463 [GenBank] ), sogS4 (EU370464 [GenBank] ), pilL1 (EU370455 [GenBank] ), pilL2 (EU370456 [GenBank] ) and pilL3 (EU370457 [GenBank] ).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Sixteen IncI1 plasmids were purified from transconjugants/transformants and analysed by RFLP by PstI restriction. They were categorized into nine different restriction patterns [A–I in Table 1 and Figure S1—available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. One strain (2392T) produced no RFLP pattern due to autolysis of the plasmid preparation.
The three available IncI1 complete DNA sequences were compared (R64-AP005147 [GenBank] , colIb-P9-AB021078 [GenBank] and pNF1358-DQ017661 [GenBank] ). Six genes were selected as potential targets for the pMLST because they marked relevant maintenance and replication plasmid functions and were well conserved but also showed significant nucleotide differences potentially useful to the subtyping of plasmids. In particular, for this analysis, we selected pilL of the cluster for the type IV pilus biogenesis, sogS, encoding the primase that acts in the discontinuous DNA plasmid replication, and ardA, encoding a type I restriction-modification enzyme. We also analysed the RNAI antisense regulating the IncI1 replication system (repI1) and the intergenic region of the trbA and pndC genes, involved in maintenance and plasmid transfer, respectively (Table 2).
The 16 IncI1 plasmids of our collection were then tested by PCR, DNA sequencing and sequence comparison with the R64 DNA sequence (AP005147 [GenBank] ). Three allele variants for repI, pilL and ardA, four alleles for sogS and six alleles for trbA-pndC were identified. Insertion of the finQ gene (encoding the fertility inhibitor) within the 5' end of the pndC gene occurred in five strains (C10-VLA, S.82/10, C1C-VLA, C12C-VLA and C13C-VLA) and characterized the trbA-pndC allele variants 5 and 6 (allele 6 was distinguished from allele 5 by 14 additional nucleotide changes in the trbA gene, Table 2).
The assortment of the different alleles defined nine different STs among the 16 IncI1 plasmids. These categories perfectly matched those obtained by RFLP (Table 1), indicating that pMLST has a comparable discriminatory power.
The three IncI1 blaCMY-2 plasmids identified in E. coli isolated from dogs in Rome (18196T) and in Padua (398T and 3115T) were assigned to the same pMLST and RFLP types (ST2, RFLP-A). They differ from the blaCMY-2 plasmid (1358T) identified by both RFLP (RFLP-B) and pMLST (ST4) in a Salmonella Thompson of human origin isolated in the USA in 1996.
The three plasmids carrying blaCTX-M-15 and blaTEM-1 genes identified in Salmonella Anatum (C1-VLA) and Typhimurium (C12-VLA and C13-VLA) in the UK were identical by both RFLP and pMLST (RFLP-C, ST8). These IncI1 plasmids were characterized by the presence of the finQ gene within the pndC gene (Table 2).
Three of the four blaCTX-M-1 plasmids were classified in the same RFLP-E and ST3 groups (Table 1). Two of them were identified in E. coli isolated from poultry in the district of Côtes d’Armor in France in 2005 (21T and 22T),7 and one was from E. coli isolated from a dog in Italy in 2005 (3960T). Interestingly, the ST3 was also assigned to a plasmid identified in E. coli isolated from poultry in Italy in the same year (2392T), but this plasmid carried the blaSHV-12 and the blaTEM-1 genes. The fourth blaCTX-M-1 plasmid (34T) was different by both restriction analysis (RFLP-D) and pMLST (ST9), and was identified in E. coli isolated from poultry in the district of Mayenne in France.7
Plasmids C10-VLA and 05-0001Tc1, carrying the blaCTX-M-14 and the blaTEM-52 genes, respectively, and plasmids R144 and S.82/10, negative for β-lactamases and showing different RFLP patterns with respect to the other IncI1 plasmids, were assigned to different STs by pMLST (ST6, ST5, ST1 and ST7, respectively; Table 1).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
The pMLST method was suitable for the rapid and easy typing of the IncI1 plasmids. The variants can be discriminated by significant and stable nucleotide divergence, matching results obtained by restriction analysis. pMLST can be applied as a second line of plasmid typing after they have been assigned to incompatibility groups by the PBRT method or other methods. A DNA sequence-based method allows recognition of similar plasmids and their detection among isolates from different countries and laboratories without exchange of strains and direct comparison of the plasmids. pMLST can also be developed for other plasmid families and can contribute to the epidemiological description of plasmid circulation in animal reservoirs and humans by describing the spread of virulence and resistance plasmids.
Our results demonstrated that indistinguishable IncI1 plasmids carrying the blaCMY-2 gene circulated in pets living in different towns in Italy in the period 2003–06, but they were different from the blaCMY-2 plasmid identified in Salmonella in the USA. Those carrying the blaCTX-M-15 gene from Salmonella isolated in the UK were identical to each other and different from the other IncI1 plasmids. Variable associations among the plasmids and the β-lactamase genes were observed: the same plasmid scaffold (ST3) was associated with different genes such as blaCTX-M-1 and blaSHV-12–blaTEM-1, and different plasmid scaffolds (ST3 and ST9) were associated with the blaCTX-M-1 gene.
From this study and the current literature, the prevalence of plasmids belonging to IncI1 seems to be linked to a particular reservoir of E. coli and Salmonella from poultry. Previous studies demonstrated that the IncI1 plasmids are significantly associated with avian pathogenic E. coli in poultry populations analysed in the USA.4 There could be a particular niche favouring these plasmids in certain specific E. coli populations, probably due to the contribution of the virulent type IV pili. This characteristic could promote the rapid spread in poultry and other food sources of the β-lactamase producers carrying IncI1 plasmids.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
This work was supported by the DRESP2 (6th PCRD, LSHM-CT-2005-018705) contract with the European Commission and the FIRB Project 'Costruzione di un laboratorio nazionale per lo studio delle resistenze batteriche agli antibiotici' contract of the Italian Ministry of Research and University.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
Figure S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Acknowledgements |
|---|
We are grateful to Dr Tonino Sofia for a critical reading of the manuscript and language revision.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsSupplementary dataReferences |
|---|
1 . 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.
2
.
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.
3
.
Kim SR, Komano T. The plasmid R64 thin pilus identified as a type IV pilus. J Bacteriol (1997) 179:3594–603.
4
.
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.
5
.
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.
6 . 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]
7
.
Girlich D, Poirel L, Carattoli A, et al. Extended-spectrum β-lactamase CTX-M-1 in Escherichia coli isolates from healthy poultry in France. Appl Environ Microbiol (2007) 73:4681–5.
8
.
Carattoli A, Lovari S, Franco A, et al. Extended-spectrum β-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003. Antimicrob Agents Chemother (2005) 49:833–5.
9
.
Couturier M, Bex F, Bergquist PL, et al. Identification and classification of bacterial plasmids. Microbiol Rev (1988) 52:375–95.
10
.
Carattoli A, Tosini F, Giles WP, et al. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998. Antimicrob Agents Chemother (2002) 46:1269–72.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Woodford, A. Carattoli, E. Karisik, A. Underwood, M. J. Ellington, and D. M. Livermore Complete Nucleotide Sequences of Plasmids pEK204, pEK499, and pEK516, Encoding CTX-M Enzymes in Three Major Escherichia coli Lineages from the United Kingdom, All Belonging to the International O25:H4-ST131 Clone Antimicrob. Agents Chemother., October 1, 2009; 53(10): 4472 - 4482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Hawkey and A. M. Jones The changing epidemiology of resistance J. Antimicrob. Chemother., September 1, 2009; 64(suppl_1): i3 - i10. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Carattoli Resistance Plasmid Families in Enterobacteriaceae Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2227 - 2238. [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Lindsey, P. J. Fedorka-Cray, J. G. Frye, and R. J. Meinersmann Inc A/C Plasmids Are Prevalent in Multidrug-Resistant Salmonella enterica Isolates Appl. Envir. Microbiol., April 1, 2009; 75(7): 1908 - 1915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sunde, H. Tharaldsen, J. S. Slettemeas, M. Norstrom, A. Carattoli, and J. Bjorland Escherichia coli of animal origin in Norway contains a blaTEM-20-carrying plasmid closely related to blaTEM-20 and blaTEM-52 plasmids from other European countries J. Antimicrob. Chemother., January 1, 2009; 63(1): 215 - 216. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||