The presence of extended-spectrum beta-lactamase (ESBL)-producing and/or AmpC-producing Salmonella and Escherichia coli in animals and food has been increasingly reported worldwide. In strains of animal origin, plasmids belonging to the Incompatibility group I1 (IncI1) have been associated with genes such as blaCTX-M-1, blaSHV-12 and blaCMY-2 [1-4]. Recently in The Netherlands, Leverstein-van Hall and colleagues demonstrated that patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains . Our previous investigation demonstrated that E. coli carrying ESBL and/or AmpC genes were genetically diverse between human and avian sources, but the potential horizontal transfer of ESBL determinants through plasmids was not investigated, and was the objective of this study .
A collection of 378 E. coli strains (277 extraintestinal pathogenic E. coli -ExPEC- strains and 101 strains from healthy avian species), previously characterized by phylogenetic typing, multilocus sequence typing (MLST) and for presence of the ESBL genes , was further analysed for the detection of the blaCMY-2 gene, and for plasmid content and localization of ESBL and AmpC genes . For plasmid comparison, six previously characterized IncI1-carrying strains were also included: two carried the blaCTX-M-1 gene, three harboured the blaCMY-2 gene and one the blaSHV-12 gene. One strain was isolated in Italy , three blaCMY-2-positive Salmonella spp. strains were isolated in the USA  (kindly provided by Dr Jason Folster, Centers for Disease Control and Prevention, Atlanta, GA, USA), and two blaCTX-M-1-positive E. coli were from France and Poland, respectively [8, 9]. Plasmid content was analysed by the PCR-based replicon typing (PBRT) method . IncI1 plasmids were further typed by plasmid multilocus sequence typing (pMLST), as previously described . Selected prototypic plasmids were further characterized by restriction fragment length polymorphism (RFLP) analysis using PstI restriction (New England Bio-Labs, Inc., Ipswich, MA, USA) . IncI1 plasmids were transferred by transformation (MAX Efficiency DH5α; Invitrogen, Milan, Italy) and transformants were screened by PBRT and for the blaCTX-M, blaSHV and blaCMY genes [6, 7, 10].
A total of 14 E. coli strains (six human isolates and eight avian isolates) possessing ESBL or AmpC genes were identified: five strains carried the blaCTX-M-1 gene, five blaCMY-2, one harboured both blaCTX-M-1 and blaCMY-2, one blaCTX-M-2 and blaCMY-2, and two strains carried the blaSHV-12 gene (Table 1). By PBRT, all 14 blaCTX-M-1-, blaSHV-12- and/or blaCMY-2-positive strains contained multiple replicons (Table 1). IncF and IncI1 plasmids were the most frequent plasmid types identified in E. coli strains of both origins and were further analysed. Transformation experiments were carried out on the 10 IncI1-IncF-positive strains, demonstrating that IncI1 plasmids were associated with the blaCTX-M-1-, blaSHV-12- and/or blaCMY-2- genes in nine of 10 human and avian strains. (Table 1).
|Strain||Origin||Country||Year||ESBL or AmpC gene||Plasmid characterization||Reference|
|PBRT in donors||PBRT in transformants||pMLSTa IncI1-plasmid||ST|
|E. coli OC144||Human||Italy||2009||bla CTX-M-1||I1, FIA, FIB, F, X1||I1||1||2||16||9||1||97||This study|
|E. coli 5c||Poultry||Italy||2009||bla CTX-M-1||I1, HI2, FIB, F||I1||1||3||3||6||2||11||This study|
|E. coli 50c||Poultry||Italy||2009||bla CTX-M-1||I1, F||I1||2||1||4||1||2||3||This study|
|E. coli OC146||Human||Italy||2009||bla CTX-M-1||FIB, F||ND||ND||This study|
|E. coli BE305||Human||Italy||2009||bla CTX-M-1||FIA, FIB, F||ND||ND||This study|
|bE. coli 37c||Poultry||Italy||2009||blaCTX-M-1, blaCMY-2||I1, N, FIB, F, K||K||–||This study|
|E. coli 41c||Poultry||Italy||2009||blaCTX-M-2, blaCMY-2||HI2, FIB, F, K, X1||ND||ND||This study|
|E. coli 65c||Poultry||Italy||2009||bla CMY-2||I1, FIB, F||I1||1||4||3||4||1||12||This study|
|E. coli 90c||Poultry||Italy||2009||bla CMY-2||I1, FIB, F, X1||I1||1||4||3||4||1||12||This study|
|E. coli BE310||Human||Italy||2009||bla CMY-2||I1, FIB, F||I1||2||4||15||11||2||85||This study|
|E. coli 44c||Poultry||Italy||2009||bla CMY-2||I1, HI2, FIB, F||I1||1||4||2||4||2||86||This study|
|E. coli PA263||Human||Italy||2009||bla CMY-2||FIB, F, K||ND||ND||This study|
|E. coli OC169||Human||Italy||2009||bla SHV-12||I1, FIB, F||I1||1||4||13||2||1||26||This study|
|E. coli 31c||Poultry||Italy||2009||bla SHV-12||I1, FIB, F||I1||2||1||4||1||2||3||This study|
|S. heidelberg AM31196||Human||USA||2007||bla CMY-2||ND||I1||1||4||3||4||1||12||3|
|S. saintpaul AM31875||Human||USA||2007||bla CMY-2||ND||I1||1||4||3||4||1||12||3|
|S. typhimurium AM33047||Human||USA||2007||bla CMY-2||ND||I1||1||4||3||4||1||12||3|
|E. coli 22T||Poultry||France||2005||bla CTX-M-1||I1||I1||2||1||4||1||2||3||8|
|E. coli 2392T||Poultry||Italy||2005||bla SHV-12||I1||I1||2||1||4||1||2||3||7|
|E. coli 96||Poultry||Poland||2009||bla CTX-M-1||I1||I1||2||1||4||1||2||3||9|
By pMLST, the nine IncI1 plasmids were classified into seven different sequence types (STs, Table 1): two on four blaCMY-2-carrying plasmids were assigned to ST12, the remaining two plasmids showed novel sequence types (ST85 and ST86). The three positively transferred blaCTX-M-1-carrying plasmids were assigned to ST3, ST11 and ST97, respectively. The two blaSHV-12-carrying plasmids were assigned to ST3 and ST26, respectively (Table 1).
The four blaCMY-2-IncI1 plasmids (two ST12 and one ST86 from poultry, and one ST85 of human origin) were compared with three blaCMY-2-IncI1/ST12 plasmids previously identified in S. enterica serovars Saintpaul, Typhimurium and Heidelberg in the USA from human sources (strains AM31196, AM31875 and AM33047), by PstI-RFLP analysis . Plasmids belonging to the same ST12 exhibited an indistinguishable profile, irrespective of both isolate species and source, while plasmids belonging to ST85 and ST86 showed different restriction profiles, as expected (Fig. 1). IncI1/ST3 and IncI1/ST97 plasmids carrying blaSHV-12 and/or blaCTX-M-1 from different sources and origins were also compared. Two blaCTX-M-1-IncI1/ST3 plasmids, both from an avian source but of different geographical origin, showed related but not identical profiles. Notably, two IncI1/ST3 plasmids carrying SHV-12, identified from poultry in Italy in 2005  and 2009 (this study), showed identical restriction profiles (Fig. 1). A different profile was exhibited by the blaCTX-M-1 -ST97 IncI1 plasmid.
Overall, ESBL-positive plasmids from our collection of E. coli appeared different according to the human or avian source, because they did not share any ST or RFLP types, even when they carried the same ESBL or CMY-2 determinant and belonged to the same Inc group.
IncI1/ST3 plasmids have been found to be associated with the blaCTX-M-1 gene among E. coli strains of avian and human origin in the Netherlands . In this study, related blaCTX-M-1-IncI1/ST3 plasmids were detected among avian strains isolated in different countries, including Italy, but not among ExPEC strains.
IncI1/ST3 plasmids harbouring blaSHV-12 were previously identified in E. coli isolates from poultry in Italy and from humans in the Netherlands [5, 7]. Our results showed persistence of an identical blaSHV-12-IncI1/ST3 plasmid over 5 years across avian isolates from Italy, but SHV-12 was found to be carried by a different plasmid in ExPEC, indicating that the epidemiology of SHV-12 on IncI1 in avian and human E. coli may differ from that described in the Netherlands, although this needs to be confirmed in additional isolates and plasmids.
Finally, IncI1/ST12 plasmids carrying blaCMY-2 were detected worldwide in E. coli and Salmonella, suggesting that the IncI1/ST12 plasmids identified in our study might be highly related to other globally diffused epidemic plasmids (http://pubmlst.org/plasmid/). This was clearly confirmed by RFLPs analysis that showed indistinguishable IncI1/ST12 plasmids obtained from both Salmonella of human origin from the USA and E. coli of an avian source from Italy, suggesting that this plasmid type can be successfully exchanged between commensal E. coli and Salmonella in poultry. However, despite the intense worldwide spread of the IncI1/ST12-CMY-2 plasmid, it was not identified in our ExPEC collection.
A limitation of this study is the low number of plasmids analysed. Such a low number makes any conclusion regarding absence of genetic relatedness among plasmids a suggestion that needs further confirmation.
In conclusion, our results suggest that, although globally diffused epidemic plasmids were identified in E. coli from an avian source from Italy, there was no evidence of common plasmids shared by both human and poultry ESBL and AmpC E. coli producers in our collection. Whether a different plasmid epidemiology actually occurs in Italy compared with other European countries needs to be confirmed by further studies.