**1. Introduction**

*Legionella* are opportunistic pathogens with a widespread distribution in freshwater environments. This bacterial genus is well known to cause legionellosis. The term "legionellosis" describes both Legionnaires' disease (LD), a severe form of atypical pneumonia, and a nonpneumonic febrile illness called Pontiac Fever. Due to the low concentrations of nutrients in their aquatic habitats, *Legionella* have adapted to live in biofilms, where they can obtain amino acids and carbon sources that they need for survival, replication and protection from temperature changes and biocide treatment [1]. In biofilms, *Legionella* are part of complex microbial communities [2] where they are subjected to predation by protozoa [3]. The transmission of bacteria from the environment to humans occurs via inhalation or aspiration of *Legionella*-containing aerosols [4,5]. Among the more than 60 species of the genus *Legionella*, *L. pneumophila* is responsible for approximately 90% of all globally reported communityand hospital-acquired cases of legionellosis [6–8]. *L. pneumophila* has 15 serogroups (Sgs); Sg1 is the most common, causing LD. Sg6 comes second, and is also a causative agen<sup>t</sup> of LD [9,10].

Many studies have demonstrated that the main sources for LD are the drinking water distribution systems (DWDS) in large buildings like hospitals and hotels [11–13]. The contamination of hospital water systems with *Legionella* is considered to pose a high risk for patients, especially for those with severe diseases. To this end, it is well known that LD is an important cause of hospital-acquired pneumonia [8]. The presence of *Legionella* in DWDS is a serious health risk to hospital staff and patients, but the magnitude of the problem is often unrecognized [8,14,15]. A high seroprevalence of *L. pneumophila* has been observed among health care workers [16]. The problem is compounded in the West Bank because awareness about the prevalence of *L. pneumophila* or LD is lacking, and few data are available for such arid regions. Furthermore, there are no specific guidelines for *L. pneumophila* surveillance or protection from exposure in hospitals or public buildings.

Cultivation Dependent Analysis (CDA) on specific agar plates is the standard and recommended technique used for environmental surveillance of *L. pneumophila* [17]. One major obstacle in the isolation and quantification of *L. pneumophila* by culture is often its Viable But Non Culturable (VBNC) state, and overgrowth by competing bacteria [18–20]. On the other hand, Cultivation Independent Analyses (CIA) using PCR-based molecular approaches are rapid, sensitive and widely applied for the detection and identification of *L. pneumophila* [21]. Because of the widespread occurrence of *L. pneumophila* in large, man-made, freshwater systems, study of environmental isolates is needed for the implementation of prevention measures, and to identify possible sources of infection [22,23].

For the identification of possible sources of contamination/infection, high resolution genotyping of new isolates is needed to correlate environmental isolates with clinical isolates. This is currently done by two molecular approaches: Sequence Based Typing (SBT) and Multilocus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA). SBT of *L. pneumophila* is done by sequencing a set of seven reference genes per isolate, providing a specific Sequence Type (ST) that can be matched with an International database [24]. MLVA has been widely used to identify different pathogens [25–27]. VNTRs consist of relatively short DNA fragments repeated in tandem that can vary in copy number among strains. For *L. pneumophila*, MLVA has exhibited an excellent ability to distinguish among strains if 8 to 12 different loci are used. It can be performed in a multiplexed manner, followed by capillary electrophoresis, enabling automated sample analysis and data acquisition. These advantages reduce typing time and costs. Several studies have used MLVA for the genotyping of *L. pneumophila* strains [28,29]. They showed the high correspondence between MLVA genotypes and STs with an important increase in resolution when applying MLVA, which is relevant for understanding clonal populations. Recent publications showed that the majority of clinically relevant strains were distributed into a limited number of Clonal Complexes (CCs) defined by MLVA, called VNTR analysis CC (VACC) and characterized by epidemic reference strains such as Paris (VACC1) and Philadelphia-1 (VACC2) [29]. Due to its advantages, MLVA could complement SBT for large sets of isolates and enable insights into the clonal structure of *L. pneumophila* populations, as well as help selecting strains for further whole genome sequencing.

The principal objective of this study was to assess the current distribution of *L. pneumophila* populations from DWDS of hospitals throughout the West Bank and determine their clonal structure and genetic diversity. To this end, *Legionella* abundances were determined for two years in bulk water and biofilms in the hospitals by applying both cultivation-dependent and -independent analyses. By the cultivation analyses, 180 *L. pneumophila* isolates were obtained from water and biofilm. These isolates were subjected to MLVA using 13 loci to reveal their clonal structure and genetic diversity. In comparison to MLVA databases, the uniqueness of the MLVA-genotypes of the West Bank could be assessed. For the West Bank, the clonal structure of *L. pneumophila* was related to the different locations and habitats and to situ-SBT analysis from pneumonia patients.

#### **2. Materials and Methods**

#### *2.1. Study Sites, Water and Biofilm Sampling*

Drinking water in the West Bank is derived from groundwater, mainly well water, with some being provided by springs. Water was provided to most of the sampled sites by the Palestinian Water Authority, except for Ramallah (sampling site D), with Mekorot as the provider. Except for site D, water treatment consisted of chlorination in storage sites before provision to the end user. All hospitals had drinking water reservoirs for water storage.

Water samples and biofilm swabs were sampled six times during the period from October 2012 to December 2014 from eight hospitals across the West Bank (Figure S1). The hospitals had the following coordinates: hospital A (coordinates: 32◦27 N, 35◦17 E), hospital B (32◦13 N, 35◦14 E) and hospital C (32◦13 N, 35◦15 E) in northern West Bank, hospital D (31◦53 N, 35◦12 E) and hospital E (31◦46 N, 35◦14 E) in central West Bank, and hospital F (31◦ 42' N, 35◦ 11' E), hospital G (31◦33 N, 35◦4 E) and hospital H (31◦31 N, 35◦5 E) in southern West Bank. Also, samples were taken from Al-Quds University (AQU) main campus, Abu Dies, East Jerusalem (31◦45 18.07 N, 35◦15 37.614 E)**.** The six samplings twice covered the main seasons, i.e., spring (March–May), summer (June–August), and autumn (October–December). It should be noted that site D could only be sampled once for spring, summer and autumn, while all other sites were sampled twice for these seasons [30].

Cold and hot water (if available) was collected from a faucet close to the hospital's drinking water reservoir and biofilm swabs were taken from faucets, showerheads, and hoses. This study is representative for Jenin, Nablus, Ramallah, Jerusalem, Bethlehem, and Hebron, going from north to south in the West Bank. The temperature, chlorine, pH, hardness, and conductivity of the water samples were determined upon collection using probes and quantofix sticks (Macherey-Nagel GmbH, Düren, Germany). Further details on sampling and individual results of the physico-chemical parameters are given in Zayed [30].
