title: Determination of N-acylhomoserine lactones of Pseudomonas aeruginosa in clinical samples from dogs with otitis externa.
abstract: BACKGROUND
  Bacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics. Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa. Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for P. aeruginosa-associated otitis externa.
  
  
  RESULTS
  Validation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs.
  
  
  CONCLUSIONS
  Our results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing.
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia. darja.kusar@vf.uni-lj.si.
authors.forename: Darja
authors.identifier: http://orcid.org/0000-0001-8583-5901
authors.initials: D
authors.lastname: Kušar
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Karin
authors.initials: K
authors.lastname: Šrimpf
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Isaković
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Lina
authors.initials: L
authors.lastname: Kalšek
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Javid
authors.initials: J
authors.lastname: Hosseini
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Irena
authors.initials: I
authors.lastname: Zdovc
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Tina
authors.initials: T
authors.lastname: Kotnik
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Modest
authors.initials: M
authors.lastname: Vengušt
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1115, Ljubljana, Slovenia.
authors.forename: Gabrijela
authors.initials: G
authors.lastname: Tavčar-Kalcher
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000382:microbiology*; D004285:Dogs; D007783:Lactones / Q000737:chemistry* / Q000378:metabolism; D010032:Otitis Externa / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007783:Lactones
keywords: Clinical samples; Dogs; Liquid chromatography-tandem mass spectrometry; N-acylhomoserine lactones; Otitis externa; Pseudomonas aeruginosa; Quorum sensing; Validation