Abstract
BackgroundBacterial 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.ResultsValidation 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.ConclusionsOur 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.
Highlights
Bacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules
With limit of detection (LOD) in the range of 100–200 pM and limit of quantification (LOQ) in 300–600 pM, LC-mass spectrometry (MS)/MS was confirmed suitable for the purpose as picomolar to micromolar concentrations were reported for clinical samples [7, 12, 22, 48, 49]
The present study demonstrated that acylhomoserine lactones (AHLs) signal molecules can be quantified in the ear rinses of Pseudomonas aeruginosa-associated OE (PaOE) affected dogs using LC-Tandem mass spectrometry (MS/MS)
Summary
Bacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. The QS system defines the expression of various bacterial phenotypes, depending on the local density of bacterial population, including the virulence factors, biofilm formation and resistance to antibiotics [11]. It is associated with pathogenesis of various diseases in humans and animals, and has a clinically evident effect in human cystic fibrosis (CF) [12], endocarditis and osteomyelitis in rabbits [13, 14], bovine mastitis [15], gastrointestinal diseases [9], and ear canal infections, dermatitis and other secondary infections in dogs [16]
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