In addition, we wanted to examine the presence of some selected genes sequences in the GPL biosynthesis gene cluster to elucidate the importance of GPLs for biofilm formation and colony morphology. To do this, the biofilm screening method needed optimisation. Methods

Eighty-eight Norwegian isolates of M. avium subspecies hominissuis from human patients Torin 2 (n = 36), swine (n = 51) and one bird and nine isolates M. avium subspecies avium originating from wild birds were examined for their ability to form biofilm (Figure 1). The isolates have been described previously [12]. In addition, the reference strains M. avium ATCC 25291, R13 and M. avium 104 were examined. M. smegmatis mc2 was included as a positive control for biofilm formation. Figure 1 Distribution of biofilm producing Mycobacterium avium isolates in a dendrogram based on the cluster analysis Apoptosis inhibitor of the composite dataset of RFLP typing using both IS 1311 and IS 1245 as probes. A total of nine isolates of M. avium subspecies avium and 88 isolates of M. avium subsp. hominissuis isolated in Norway were included. The RFLP dendrogram has been presented elsewhere [12], but is has presently been combined with additional information regarding hsp65 code and the biofilm forming abilities of the isolates.

#1247 represents the identical profiles of nine avian isolates, including #1553 and #1794. Biofilm forming isolates have been highlighted in pink. 3-mercaptopyruvate sulfurtransferase Method optimisation A panel of 14 M. avium subsp. hominissuis (seven from humans, six from swine and one from a bird), and three M. avium subsp. avium isolates originating from birds, including the reference strains

ATCC 25291 and R13, and the positive control M. smegmatis mc2 were used during optimisation of the method. The isolates all had a low passage number. Biofilm formation was determined as previously described [30], but with some modifications. Isolates were cultured in 10 ml Middlebrook 7H9 (BD Diagnostics, Sparks, MD) containing 10% oleic acid, albumin, dextrose and catalase (BD Diagnostics), 0.1% Tween 80 (Merck KGaA, Darmstadt, Germany) and 0.2% glycerin (Merck) (7H9 with OADC and Tween). They were incubated with agitation (100/min) at 37°C for minimum two weeks until they reached the stationary phase at which point Cell Cycle inhibitor culture aliquots were frozen at -70°C. Subsequently, 100 μl of frozen stock culture was inoculated in 10 ml of fresh 7H9 with OADC and Tween and incubated at 37°C with agitation for seven days. OD600 was measured, and the cultures were centrifuged and resuspended to an OD600 of 0.2 in the different medias described below. 200 μl of the cell suspension were added to the wells of a 96-well flat bottom polystyrene microtiter plate in triplicates (MicroWell™ Plates Nunclon™Δ no.