Dissemination of blaTEM-52 among Escherichia coli and Klebsiella pneumoniae strains is caused by spread of plasmids in Portugal A. Pedrosa 1, 2 , A. Novais 2 , E. Macha<strong>do</strong> 1,3 , R. Cantón 2 , T. M. Coque 2 *, L. Peixe 1 1 Department of Microbiology, Faculty of Pharmacy, University of <strong>Porto</strong>, Portugal. 2 Hospital Universitario Ramón y Cajal, IMSALUD, Madrid, Spain 3 Faculty of Ciências da Saúde, Fernan<strong>do</strong> Pessoa University, <strong>Porto</strong>, Portugal TEM-52 was firstly described in 1996 and since then it has been sporadically identified in Europe, Korea and Canada among Enterobacteriaceae from both human and animals [1, 2]. Recently, an epidemic IncI1 plasmid containing blaTEM-52 was identified in France among Salmonella enterica community strains[2]. We analyzed the diversity of TEM-52 producing isolates from Portugal in order to understand the reasons for recent spread in our country. Twenty one TEM-52-producing isolates recovered from Portugal (n=19 Escherichia coli; n=2 Klebsiella pneumoniae) between 2002 and 2005 were studied. The isolates were obtained from hospitals (n=7), poultry (n=12) and sewage (n=2). Only one phenotype was included. Clonality was established by PFGE and E. coli phylogenetic groups by PCR as reported [3, 4]. ESBL identification was achieved by IEF, PCR and sequencing. Standard methods were used for conjugation assays as well as antibiotic susceptibility testing. bla location was addressed by hybridization of I-CeuI-digested DNA with an intragenic blaTEM-52 probe [5]. Plasmid characterisation was accomplished by the Barton’s method, identification of plasmid incompatibility groups by PCR, hybridization and sequencing, and analysis of RFLP patterns [6, 7]. The isolates studied were highly diverse and 21 PFGE-types were identified [3]. Most E. coli recovered from clinical isolates were assigned to phylogenetic group B1 [4]. E. coli recovered from poultry and sewage were identified as phylogroups A (4 A0 and 3 A1), B1 (n=2) and D (3 D1 and 1 D2). Although chromosomal location was identified in one strain, blaTEM-52 gene was mostly carried by two different plasmids of approximately 90kb and 30kb [1, 5]. The 90kb plasmids belong to the incompatibility groups I1 but the 30kb could not be typed by the PCRtyping method used. All the 30kb plasmids presented a unique RFLP pattern and most of the 90kb plasmids had the same RFLP pattern. Wide and recent dissemination of blaTEM-52 among E. coli and K. pneumoniae strains from Portugal seems to be associated with dissemination of particular plasmids of 30 and 90kb among highly unrelated E. coli and K.pneunoniae strains. [1] Weill, F., Demartin, M., Fabre, L. and Grimont, P.A.D. (2004), Extended-Spectrum-β-lactamase (TEM- 52)-Producing Strains of Salmonella enterica of Various Serotypes Isolated in France, Journal of Clinical Microbiology, 42(7):3359-3362. [2] Cloeckaert, A., Praud, K., Doublet, B., Bertini, A., Carattoli, A., Butaye, P., Imberechts, H., Bertrand, ., Collard, J., Arlet, G. and Weill, F. (2007), 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, Antimicrobial Agents and Chemotherapy, 51(5):1872-1875. [3] Liu, S.-L., and Sanderson, K. E. (1992), A physical map of the Salmonella typhimurium LT2 genome made by using XbaI analysis, Journal of Bacteriology, 174(5):1662-1672. [4] Clemont, O., Bonacorsi, S. and Bingen, E. (2000), Rapid and Simple Determination of the Escherichia coli Phylogenetic group, Applied and Environmental Microbiology, 66(10):4555-4558. [5] Liu, S.-L., Hessen, A. and Sanderson, K. E. (1993), Genomic mapping with I-Ceu I, an intron-encoded en<strong>do</strong>nuclease specific for genes for ribosomal RNA, in Samonella spp., Escherichia coli, and other bacteria, Proc. Natl. Acad. Sci. USA, 90:6874-6878. [6] Barton, B., Harding, G. and Zuccarelli, A. J. (1995), A General Method for Detecting and Sizing Plasmids, Analytical Biochemistry, 226:235-240. [7] Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K. L. and Threlfall, J. (2005), Identification of plasmids by PCR-based replicon typing, Journal of Microbiological Methods, 63:219-228. 25
Synergism between adenosine A2A and tachykinin NK1 receptors facilitate [3H]-ACh release from myenteric neurons I. Silva, C. Vieira, M. Duarte-Araújo & P. Correia-de-Sá Laboratório de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigação Biomédica (UMIB), Instituto de Ciências Biomédicas Abel Salazar - <strong>Universidade</strong> <strong>do</strong> <strong>Porto</strong> (ICBAS-UP), Portugal. En<strong>do</strong>genous tachykinins, such as substance P (SP) and neurokinin A (NKA), are expressed in distinct neural pathways of the mammalian gut and have the potential to control both nerve and muscle activity [1]. In addition to inhibitory adenosine A1 receptors expressed on both cholinergic and tachykinergic myenteric neurons [2], activation of prejunctional A2A receptors facilitate acetylcholine (ACh) release from myenteric neurons [3]. Therefore, we decided to investigate the influence of en<strong>do</strong>genous adenosine on tachykinin-mediated facilitation of [ 3 H]-ACh release from longitudinal muscle-myenteric plexus (LM-MP) preparations of the rat ileum. The experiments were performed at 37ºC on LM-MP preparations from control subjects (C) and from animals injected subcutaneously with capsaicin in the neonatal period (CAP). Neonatal capsaicin causes degeneration of peptidergic nerve fibres with loss of sensation and smooth muscle contractility. Procedures used for labeling the preparations and measuring evoked [ 3 H]-ACh release were previously described [3]. Facilitation of [ 3 H]-ACh release caused by the selective A2A receptor agonist CGS 21680C (3 nM, 53±10%, n=5) was significantly (P
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