title: Biofilm formation and susceptibility to gentamicin and colistin of extremely drug-resistant KPC-producing Klebsiella pneumoniae.
abstract: OBJECTIVE
  KPC-producing Klebsiella pneumoniae (KPC-Kpn) is a worldwide challenging pathogen, yet its biofilm-forming potential is not defined. We characterized biofilm formation of this pathogen and determined biofilm susceptibility to gentamicin and colistin.
  
  
  METHODS
  Forty-six KPC-Kpn clinical isolates were studied [sequence type (ST) 258, n = 28; and other STs, n = 18]. Biofilm biomass was determined using the standard assay measured by OD590 (where OD stands for optical density) and visualized using confocal microscopy. Antibiotic effect on biofilm formation was evaluated and susceptibility within biofilm was determined by the minimal biofilm elimination concentration (MBEC) method.
  
  
  RESULTS
  KPC-Kpn isolates produced biofilm in the range of 0.02-0.3 OD590, where ST258 isolates produced less biofilm compared with other STs (median OD590 0.07 versus 0.15, respectively; P < 0.05). Biofilm biovolumes were in the range of 354 ± 323 to 27,461.4 ± 11,886.7 μm(3). In the planktonic state, ST258 isolates were less resistant to gentamicin compared with other STs (resistance rates: 14% versus 66%, respectively; P < 0.05). Gentamicin-resistant isolates (MIC ≥ 32 mg/L) showed a dramatic increase in resistance within the biofilm (up to 234-fold), whereas gentamicin-susceptible isolates (MIC <32 mg/L) retained their susceptibility. The elevated gentamicin resistance was not due to overexpression of the aminoglycoside resistance gene aac(3)-II in the biofilm state. Resistance to colistin in biofilm increased as well, but was less prominent (P < 0.05). Biofilm biomass did not affect the MBECs of gentamicin and colistin, regardless of the genetic lineage.
  
  
  CONCLUSIONS
  KPC-Kpn and particularly ST258 do not form massive biofilms. Nevertheless, susceptibility to gentamicin of this endemic lineage is retained in its biofilm state, supporting the use of this antibiotic in the clinical scenario.
authors.affiliation: Molecular Epidemiology and Antimicrobial Resistance Laboratory, Division of Epidemiology, Tel Aviv Medical Center, Tel Aviv, Israel.
authors.forename: Livnat
authors.initials: L
authors.lastname: Naparstek
authors.forename: Yehuda
authors.initials: Y
authors.lastname: Carmeli
authors.forename: Shiri
authors.initials: S
authors.lastname: Navon-Venezia
authors.forename: Ehud
authors.initials: E
authors.lastname: Banin
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018441:Biofilms / Q000254:growth & development*; D003091:Colistin / Q000494:pharmacology*; D005839:Gentamicins / Q000494:pharmacology*; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D001618:beta-Lactamases; D003091:Colistin
keywords: K. pneumoniae; MBECs; biofilm resistance; carbapenem resistance; sequence type 258