title: Genetically unrelated multidrug- and carbapenem-resistant Citrobacter freundii detected in outpatients admitted to a Portuguese hospital.
abstract: Non-clonal, carbapenem- and multidrug-resistant Citrobacter freundii isolates were collected from unrelated outpatients admitted to a Portuguese hospital emergency department. One patient lived in a nursing home and was never hospitalised, whereas the other patient was repeatedly hospitalised in this hospital. The aim of this study was to unveil the molecular mechanisms associated with the carbapenem resistance of these isolates and to assess its potential dissemination.
  
  
  
  Isolate identification was performed by VITEK®2 and was confirmed by 16S rDNA sequencing. The clonal relationship of the isolates was evaluated by repetitive element palindromic PCR (rep-PCR). Antibiotic susceptibility was determined using the automatic VITEK®2 AES system and disk diffusion assay. β-Lactamases, porins and mobile genetic elements were characterised by PCR and sequencing. Pulsed-field gel electrophoresis (PFGE) and Southern blot hybridisation were used to determine the genetic location of integrons, and their transferability was tested by conjugation.
  
  
  
  No genetic relatedness was found, suggesting different origins of the isolates. In isolate Cf254 a VIM-2 carbapenemase integrated in In58 was detected, located in a high-frequency conjugative IncL/M plasmid that also carried CTX-M-15 and CMY-39 genes. VIM-1 in isolate Cf872 was chromosomal. This is the first description in Portugal of VIM carbapenemases in C. freundii. Loss/alteration of porins was also detected.
  
  
  
  Emergence of carbapenem-resistant Enterobacteria is not confined to the nosocomial environment. Community-acquired strains appear to be in circulation between inpatients and outpatients, spreading carbapenem resistance genes by horizontal gene transfer.
authors.affiliation: CESAM and Department of Biology, University of Aveiro, Campus de Santiago, 3810 Aveiro, Portugal. Electronic address: csantos.bio@gmail.com.
authors.forename: Cátia
authors.initials: C
authors.lastname: Santos
authors.affiliation: Centro Hospitalar do Baixo Vouga, Avenida Artur Ravara, 3810 Aveiro, Portugal.
authors.forename: Elmano
authors.initials: E
authors.lastname: Ramalheira
authors.affiliation: Faculty of Pharmacy and Centre for Neurosciences and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Da Silva
authors.affiliation: CESAM and Department of Biology, University of Aveiro, Campus de Santiago, 3810 Aveiro, Portugal.
authors.forename: Sónia
authors.initials: S
authors.lastname: Mendo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015780:Carbapenems / Q000494:pharmacology*; D016971:Citrobacter freundii / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D016172:DNA Fingerprinting; D052978:Disk Diffusion Antimicrobial Tests; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics*; D004755:Enterobacteriaceae / Q000235:genetics; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial / Q000235:genetics*; D006761:Hospitals; D006801:Humans; D032023:Integrons / Q000235:genetics; D020071:Interspersed Repetitive Sequences / Q000235:genetics; D017720:Molecular Epidemiology; D058889:Molecular Typing; D010045:Outpatients; D011174:Portugal; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D012336:RNA, Ribosomal, 16S; C473109:beta-lactamase CTX-M-15; C425108:beta-lactamase bla(vim-2); D001618:beta-Lactamases; C063912:carbapenemase
keywords: Carbapenem resistance; Community; Conjugative plasmids; Multidrug-resistant Citrobacter; Outpatients; VIM and CMY enzymes