*2.3. Phylogenetic Analysis*

Phylogenetic analysis using SBT of a subset of 78 isolates revealed 27 different STs, including two novel STs (ST2169, ST2284), with the index of diversity being 0.912. Twelve STs were associated with more than one isolate, and 16 STs were identified with one single isolate. The most prevalent STs found were ST1 (26%), ST87 (10%), ST93 (6%), and ST461 and ST1516 (5% each). Of all Lp sg1 isolates, ST1 accounted for 63% (20/32), while the leading Lp non-sg1 subtype, ST87, comprised 17% of all sg2–14 isolates (8/46). Amplification failure of the *flaA* fragment occurred in two allelic

profiles (0,4,16,1,7,13,206 and 0,14,16,1,7,13,206), and therefore no STs could be obtained for them. The clustering analysis of the 27 strains is shown in Figure 2.


**Figure 2.** Similarity dendrogram of 78 *L. pneumophila* strains isolated from hotel and resort water systems in Israel. A phylogenetic tree was constructed using unweighted pair group method with arithmetic averages (UPGMA) clustering. The sequence type (ST), serogroup, source by category type, and region of isolation are indicated. The asterisks indicate untypeable isolates (failed *flaA* gene amplification).

While several *L. pneumophila* sequence types were distributed widely throughout the country (ST1, ST1642, and ST461), a number of strains have been found to be limited to certain geographical regions. Specifically, ST59, ST1326, and ST1641 were unique to the Jerusalem district, and ST1516 was only found in the Southern region. Moreover, the Southern and Jerusalem districts displayed the most diverse *L. pneumophila* population, with 11 and 10 different STs, respectively (Figure 3).

**Figure 3.** Minimum spanning tree (MST) based on sequence-based typing (SBT) profiles of 78 Lp isolates from hotel and resort water systems. Sequence type (ST) is indicated next to the circles, circle size is proportional to the number of isolates sharing the same ST, and each isolate is shown as a segmen<sup>t</sup> of the relevant circle. Branches connect the STs and show the genetic distance between them. STs differing by five or more alleles are not connected. Color-coding of the circles denotes geographic region. Nodes without an ST number represent strains closely related to ST1334 with failed *flaA* typing.
