**Characterization of** *Legionella pneumophila* **Populations by Multilocus Variable Number of Tandem Repeats (MLVA) Genotyping from Drinking Water and Biofilm in Hospitals from Di**ff**erent Regions of the West Bank**

#### **Ashraf R. Zayed 1,2, Marina Pecellin 1, Alaa Salah 2, Hanna Alalam 2, Suha Butmeh 2, Michael Steinert 3, Rene Lesnik 1, Ingrid Brettar 1, Manfred G. Höfle 1,\* and Dina M. Bitar 2**


Received: 30 September 2020; Accepted: 19 October 2020; Published: 22 October 2020

**Abstract:** The West Bank can be considered a high-risk area for Legionnaires' disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. *Legionella*, mostly *L. pneumophila*, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of *Legionella* spp and *L. pneumophila* using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of *Legionella* in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 *L. pneumophila* strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of *L. pneumophila.* Overall, our results provide important insights into the detailed population structure of *L. pneumophila*, allowing for better risk assessment for DWDS.

**Keywords:** MLVA-genotypes; clonal complex; hospital water; West Bank; *Legionella pneumophila*
