b + indicates pks15/1 gene intact. c -indicates absence of the RD105 genomic region. By origin, 22 of the 26 isolates were from foreign-born cases (84.6%) of nine different nationalities, the most frequent being Peruvians and Ecuadorians (42%). The remaining four Beijing isolates corresponded to autochthonous cases (Table 1).

The drug susceptibility tests showed that 23 of the 26 isolates were pan-susceptible, two were isoniazid-resistant, and one was multidrug-resistant (Table 1). Genotyping analysis The IS6110-RFLP analysis revealed 21 different genotypes (9-22 IS6110 copies). Seven isolates (26.9%) were grouped in two clusters of three and four cases each. Nineteen isolates (73.1%) were unclustered and considered orphan cases (Figure 1A). The isolates involved in cluster 2 (C2) shared an identical IS6110-RFLP pattern Ibrutinib with those involved in the Gran Canaria outbreak [14]. Figure 1 Comparative analysis of IS 6110 -RFLP (A), MIRU-15 (B), and MIRU-15+5 (C) in the 26 clinical Beijing isolates. aOrder of QUB loci: QUB 11a, QUB 3232, and QUB 18. bOrder of VNTR loci: VNTR3820 and VNTR4120. The clustered cases are indicated within boxes. C1 and C2 refer to the cases included in the two clusters defined by RFLP. In some cases, the large

size of some products obtained in QUB and the VNTR loci did not allow precise assignation of alleles. In these cases we could only estimate that the

number of repetitions was higher than 20 (> 20). Y-27632 datasheet When we observed products differing in size in groups of isolates with more than Cyclic nucleotide phosphodiesterase 20 repetitions, we sub-labeled them > 20a, > 20b, > 20c and > 20d. The MIRU-15 analysis identified 18 different genotypes among the Beijing isolates. Thirteen isolates (50%) were grouped in five clusters of two or three cases. The remaining isolates corresponded to orphan cases (Figure 1B). If we compare RFLP and MIRU-15 data, it is noteworthy that two representatives of cluster 1 (C1), defined by RFLP, were split by MIRU-15, and three of the clusters defined by MIRU-15 grouped isolates that had been considered orphan by RFLP. Only the C2 cluster defined by RFLP remained intact after MIRU analysis. Regarding the isolates clustered in C2, which shared the RFLP pattern with the isolate involved in the Gran Canaria outbreak, we also pursued to compare the MIRU-15 data. With this aim, a selection of Gran Canaria outbreak isolates, sharing also the susceptibility pattern with those form Madrid, were analyzed and an identical MIRU-15 type was shared by the representatives from Madrid and Gran Canaria. After observing the low discrimination of MIRU-15, five new VNTR loci (QUB11a, QUB3232, QUB18, VNTR3820, and VNTR4120) were added; they were all selected due to their high discriminatory values in different studies focused on Beijing isolates [19, 20].