Enterobacteriaceae resistant to extended-spectrum β- lactam antibiotics in different settings in Portugal

The expansion of extended-spectrum β-lactamases (ESBLs) and carbapenemases in both nosocomial and community settings has seriously compromised the use of βlactam antibiotics in the treatment of infections caused by Enterobacteriaceae. In Portugal, a high occurrence of ESBLs has been reported and carbapenemase-producing isolates have recently emerged, although recent epidemiological data on the occurrence, diversity, and epidemiological features of ESBLor carbapenemase-producing Enterobacteriaceae are scarce. The global goal of this study is the multi-level molecular epidemiological characterization of recent Enterobacteriaceae isolates (2006-2010) resistant to extended-spectrum cephalosporins and/or carbapenems obtained from different ecological niches (hospitalized patients, pig farms). Three hundred and two Enterobacteriaceae isolates obtained from different sources (5 hospitals, 2 piggeries) between 2006 and 2010 were studied. They included i) 264 ESBL-producing isolates from 3 hospitals; ii) 16 isolates with reduced susceptibility to carbapenems from 2 hospitals; and iii) 22 ESBL-producing isolates from animals, feed and environmental samples of two Portuguese piggeries. Bacterial identification and antibiotic susceptibility testing were performed by standard methods. ESBL or carbapenemase genes and their genetic environment were identified by PCR (blaCTX-M, blaSHV, blaTEM, blaKPC, blaMBL, blaOXA) and sequencing. Clonal relatedness was established by XbaI-PFGE and MLST. Plasmid analysis included identification of incompatibility groups by PCR, sequencing and hybridization, RFLP and pMLST. Porin changes were investigated by PCR and SDS-PAGE. Resistance to extended-spectrum cephalosporins was mostly observed in Escherichia coli and Klebsiella pneumoniae, and also Enterobacter cloacae, Klebsiella oxytoca, Proteus mirabilis and Serratia marcescens. A high diversity of ESBLs was detected, mostly from CTX-M (CTX-M-1, -2, -9, -14, -15, -32, -79) and SHV-types (SHV-2, -5, -12, -28, -55, -106, -145), particularly among E. coli and K. pneumoniae isolates, respectively, while the TEM-type enzymes were sporadically reported (TEM10, -24, -52, -116, -199) in different Enterobacteriaceae species. Despite the high clonal diversity observed (264 isolates/86 PFGE-types), a few epidemic E. coli (ST131) and K. pneumoniae (ST15, ST147, ST336) clones were associated with the dissemination of particular ESBL-types (CTX-M-15, different SHV-variants) during large periods of time in different hospitals. CTX-M-1/-32 or TEM-52 types were detected among Portuguese piggeries associated with the spread of epidemic plasmids (IncI1/ST3 and IncN, respectively) and clones (E. coli clonal complex 10), frequently identified in humans and other animals in several countries. Reduced susceptibility to carbapenems was detected: i) in a nosocomial outbreak involving different ESBL or plasmidmediated AmpC-producing Enterobacteriaceae isolates exhibiting porin alterations; and ii) in a ST15 K. pneumoniae clinical isolate producing a novel VIM-1 variant, designated VIM-34. A complex epidemiology of ESBL-producing Enterobactericeae in Portugal is described, with the involvement of a diversity of ESBL-types, epidemic clones (in some cases harbouring closely related blaESBL genes) and plasmids. The identification of identical ESBL-encoding plasmids and clones between isolates from human and animal origins stresses a link through the food chain. The detection of strains with decreased susceptibility to carbapenems (either by carbapenemase production or ESBL/AmpC production plus porin changes) belonging to widespread clones in Portuguese hospitals was important to implement appropriate and timely infection control measures, and to reinforce continuous surveillance systems. Enterobacteriaceae resistant to extended-spectrum βlactam antibiotics in different settings in Portugal: towards an epidemiological characterization of a Public Health priority This work was financially supported by research grants from: • Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior of Portugal (grants numbers PEst-C/EQB/ LA0006/2011 and POCI/AMB/61814/2004-FSE/FEDER • Fundação Ensino e Cultura Fernando Pessoa • Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (grant number PIEF-GA-2009-255512 to Ângela Novais) • REQUIMTE (Laboratório Associado) Aos meus pais, Por tudo o que são para mim. “O único lugar onde o sucesso vem antes do trabalho é no dicionário. ” Albert Einstein