Degradation of mucin

The capacity of the 49 pre-selected A-1155463 in vitro LAB to degrade gastric mucin was determined as described by Zhou et al.[58]. Mucin from porcine stomach type III (Sigma-Aldrich Corp.) and agar were added to medium B without glucose at concentrations of 0.5% (w/v) and 1.5% (w/v), respectively. A volume of 10 μl of 24 h viable bacterial cultures was inoculated onto the surface of medium B. The plates were incubated anaerobically at 37°C for 72 h, subsequently stained with 0.1% (w/v) amido black (Merck KGaA) in 3.5 M acetic acid for 30 min, and then washed with 1.2 M acetic acid (Merck KGaA). A discoloured zone around the colony was considered as a positive result. A fresh fecal slurry of a healthy adult horse was used as positive control for mucin degradation ability. Determination of enzymatic activities The APIZYM test (BioMérieux, Montallieu Vercieu, France) was used for determination of enzymatic activities of the 49 pre-selected LAB. Cells from cultures grown at 32°C overnight were harvested check details by centrifugation

at 12,000 g for 2 min, resuspended in 2 ml of API Suspension Medium (BioMérieux) and adjusted to a turbidity of 5–6 in the McFarland scale (approx. 1.5-1.9 × 109 CFU/ml). Aliquots of 65 μl of the suspensions were added to each of the 20 reaction cupules in the APIZYM strip. The strips were incubated at 37°C for 4.5 h and the reactions were developed by addition of one drop each of the APIZYM reagents A and B. Enzymatic activities were graded from 0 to 5, and converted to nanomoles as indicated by the manufacturer´ s instructions. PCR detection of antibiotic resistance genes The presence of genetic determinants conferring resistance to aminoglycosides except streptomycin aac(6´)-Ie-aph(2´´)-Ia, to erythromycin erm(A), erm(B), erm(C) and mef(A/E)], to tetracycline tet(K), tet(L) and tet(M)], and to lincosamides lnu(A) and lnu(B)] was determined by PCR in the LAB strains showing antibiotic resistance by the VetMIC assay. PCR-amplifications

and PCR-product visualization and analysis were performed as described above using the following primer-pairs: aacF/aacR for detection of aac(6´)-Ie-aph(2´´)-Ia[74], ermAI/ermAII for erm(A) [75, 76], ermBI/ermBII for erm(B) [17], ermCI/ermCII for erm(C) Farnesyltransferase [17, 77], mef(A/E)I/ mef(A/E)II for mef(A/E) [75, 76], tetKI/ tetKII for tet(K) [17], tetLI/tetLII for tet(L) [17, 78], tetMI/tetMII for tet(M) [17, 78], lnuA1/lnuA2 for lnu(A) [79], lnuB1/lnuB2 for lnu(B) [50]. E. faecalis C1570 was used as positive control for amplification of erm(C), lnu(A) and tet(K) and E. faecalis C1231 for erm(A). E. faecium 3Er1 (Tipifarnib concentration clonal complex of hospital-associated strain CC9) and E. faecium RC714 were used as positive controls for amplification of aac(6´)-Ie-aph(2´´)-Ia, tet(M) and tet(L), and for erm(B) and mef(A/E), respectively.