Api kits

All chemicals and solvents were of the analytical and chromatographic grade. Standard biogenic amines (agmatine sulfate, tryptamine hydrochloride, 2-phenylethylamine, putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tyramine hydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride), dansyl chloride, and acetone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide, sodium hydrogen carbonate, ammonium hydroxide, and perchloric acid were purchased from Junsei Chemicals (Tokyo, Japan). High-performance liquid chromatography (HPLC) grade acetonitrile and ammonium acetate were obtained from Merck (Darmstadt, Germany). Nutrient agar was purchased from Difco (Sparks, MD, USA). API kits were purchased from BioMerieux (Marcy I’ Etoile, France).

Serial two-fold dilutions of the KB were made; then, 0.1 mL aliquots from these dilutions were pipetted onto MRS agar plates [77 ]; specific Acetobacter agar (Oxoid, UK); Sabaraoud agar (Oxoid) for isolation of lactic acid bacteria; acetic acid bacteria; yeasts, respectively. The agar plates were incubated at 35 °C for either 48 h for bacteria or 4 days for yeasts. Pure and single colonies of the obtained microbes were picked up by sterile needles and inoculated into Brain Heart Infusion broths (BHI broth, Oxoid). After 24 h of incubation at 35 °C, 100 µL aliquots of microbial suspensions were loaded aseptically onto API kits (BioMérieux, France) that were then used for identification of the microorganisms isolated as given by the manufacturer’s instructions. Both bacteria and fungi isolated were checked for their Gram stain and cell morphology using a light microscope [2 ,3 (link),78 ,79 ].

Gastric juice, sputum samples and PEG-s underwent microbiological culture performed in accordance with national accredited methods. 10 μl of homogenised sputum, gastric juice and PEG-conditioned saline were plated for bacterial and fungal cultures. The following media were used: Columbia blood agar supplemented with 5% horse blood, chocolate agar supplemented with 70 mg/L bacitracin, Burkholderia selective agar (for CF sputa and gastric juice, CEP bioMérieux UK), cysteine lactose electrolyte deficient agar (CLED) and fastidious anaerobic agar (FAA). Plates were incubated according to a standard protocol, CEP cultures were incubated for 10 days at 30 °C for isolation of Burkholderia cepacia complex and rapidly growing mycobacterium.
Plates were examined daily for evidence of microbial growth and assessment of the number of distinct colonial variants was recorded. All morphological variants were sub-cultured and used for identification and stored at −20 °C in 10% glycerol skimmed milk. Isolates were identified by matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, UK) and where necessary, appropriate API kits (bioMérieux, UK)15 (link). Mycobacterium was identified by rpoB, sodA and hsp65 gene sequencing and strain typed using variable number tandem repeat (VNTR), Colindale, UK7 .