Luisa Peixe

To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates (n 21) and genome or plasmid sequences (n 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates , the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chro-mosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobac-ter isolates revealed that each platform evolved in different recognizable (Citrobacter freundii, C. braakii, C. werkmanii, and C. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae. Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers.

Objectives: The aim of this study was to assess the diversity of Enterococcus faecalis populations recovered in different regions of Portugal during the last decade (1996– 2007) and to analyse their genetic elements associated with vancomycin resistance. Methods: Forty E. faecalis isolates (22 vancomycin-susceptible and 18 vancomycin-resistant) representing disseminated and/or multiresistant strains from different sources (humans, animals and the environment) were characterized by PFGE and multilocus sequence typing. Genes encoding putative virulence markers and the backbone of Tn1546 were investigated by PCR. Plasmid analysis included determination of size, content (S1 hybridization) and comparison of restriction fragment length poly-morphism patterns. Results: The 40 E. faecalis isolates (22 PFGE types) mostly clustered within the worldwide-spread clonal complexes (CCs) CC2 (13 ST6 mostly corresponding to an epidemic strain, where ST stands for sequence type), CC21 (3 ST21, 1 ST22 and 1 ST224) and ST16 (n 57), but also comprised CC21 were isolated from both hospital and community settings. Similar Tn1546-like elements encoding VanA were found on related plasmids within strains belonging to different clonal lineages and recovered in distinct hospitals over several years. Conclusions: The predominance of E. faecalis CC2 is mainly due to the dissemination of a particular clone persistently recovered for 11 years. The presence in the community of specific strains belonging to major clonal lineages highlights the role of community-associated hosts as possible reservoirs of putative human pathogenic enterococci. Both clonal expansion and dissemination of epidemic conju-gative VanA plasmids seem to join forces in the establishment of pathogenic E. faecalis strains.

Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumptively associated with the expansion of a human-associated Enterococcus faecium polyclonal subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin and vancomycin) and virulence (esp Efm , hyl Efm , and fms) traits. We analyzed the presence and the location of a putative glycoside hydrolase hyl Efm gene among E. faecium strains obtained from hospitalized patients (255 patients; outbreak, bacteremic, and/or disseminated isolates from 23 countries and five continents; 1986 to 2009) and from nonclinical origins (isolates obtained from healthy humans [25 isolates], poultry [30], swine [90], and the environment [55]; 1999 to 2007). Clonal relatedness was established by pulsed-field gel electro-phoresis (PFGE) and multilocus sequence typing (MLST). Plasmid analysis included determination of content and size (S1-PFGE), transferability (filter mating), screening of Rep initiator proteins (PCR), and location of vanA, vanB, ermB, and hyl Efm genes (S1/I-CeuI hybridization). Most E. faecium isolates contained large plasmids (>150 kb) and showed variable contents of van, hyl Efm , or esp Efm. The hyl Efm gene was associated with megaplasmids (170 to 375 kb) of worldwide spread (ST16, ST17, and ST18) or locally predominant (ST192, ST203, ST280, and ST412) ampicillin-resistant CC17 clones collected in the five continents since the early 1990s. All but one hyl Efm-positive isolate belonged to the CC17 polyclonal subcluster. The presence of hyl Efm megaplasmids among CC17 from Europe, Australia, Asia, and Africa since at least the mid-1990s was documented. This study further demonstrates the pandemic expansion of particular CC17 clones before acquisition of vancomycin resistance and putative virulence traits and describes the presence of megaplasmids in most of the contemporary E. faecium isolates with different origins.

TEM-154, identified in Portugal in 2004, associated the substitutions observed in the extended-spectrum-lactamase (ESBL) TEM-12 and in the inhibitor-resistant penicillinase (IRT) TEM-33. This enzyme exhibited hydrolytic activity against ceftazidime and a low level of resistance to clavulanic acid. Surprisingly, the substitution Met69Leu enhanced the catalytic efficiency of oxyimino-lactams conferred by the substitution Arg164Ser. Its discovery confirms the dissemination of the complex mutant group of TEM enzymes in European countries.

ABSTRACT  Aim: To characterize temporal shifts in extended-spectrum β-lactamases (ESBLs) and clones of clinical Escherichia coli isolates. All ESBL-producing E. coli isolates from a Portuguese hospital (n = 112; June 2006-June 2007 and January-December 2010) were characterized by identification of phylogenetic groups, ESBL-types and virulence genes, XbaI-PFGE and MLST. We observed a substantial increase in widespread E. coli clones from phylogroups A, B1 and D (e.g., ST10, ST23, ST117, ST155, ST648) producing mainly CTX-M-1, -14, -32 or SHV-12, along with a decrease in the proportion of the predominant CTX-M-15-producing B2-ST131 clone. The amplification of diverse CTX-M-producing A, B1 and D clonal complexes, which have been long identified in Portuguese nonclinical settings, unveils a role for these reservoirs in the landscape of ESBL-producing E. coli in the clinical setting.