**2. Results**

One thousand two hundred and seventy abstracts were obtained from the Web of Science and SCOPUS. After applying the described criteria (see Figure 1 and the Materials and Methods section), 29 research manuscripts discussing *L. pneumophila* and its protozoan hosts in hospital and potable water systems were included in the study (Table 1). Potential protozoan hosts playing crucial role(s) in the *L. pneumophila* life-cycle and living in both types of water systems are compiled in Table 2. These protozoan hosts have the potential to provide an appropriate habitat for replication and survival of *L. pneumophila*.

The articles from hospital settings showed that *L. pneumophila* Serogroup 1 (hereafter SG1) is the most common serogroup causing infection in USA and European countries. Globally, SG1 is also associated with community acquired legionellosis [29,30]. However, a limitation was that most municipal potable water supply studies did not characterize the *L. pneumophila* serogroups. To investigate the different *L. pneumophila*-protozoan interactions, some studies used co-isolation and co-culturing techniques or PCR. Other approaches included techniques like scanning electron microscopy or DVC-FISH to demonstrate the fate of internalized bacteria. The electron microscope studies conducted in hospital settings found that *L. pneumophila* SG1 is able to multiply inside *Echinamoeba exudans* [31] and *Vermamoeba vermiformis* (formerly *Hartmannella vermiformis*) [32]. Likewise, PCR-based examination of potable

water also demonstrated the presence of *L. pneumophila* inside *V. vermiformis* [33]. Another study used DVC-FISH to detect intracellular *L. pneumophila* inside *Acanthamoeba* and *V. vermiformis* from a potable water supply [34]. Other studies (mentioned in the Table 1), demonstrated the co-existence of free-living *L. pneumophila* and protozoan hosts, but did not characterize the specific interaction or fate of internalized bacteria. The systematic literature review identified a more diverse number of potential protozoan hosts from hospitals compared with municipal potable water systems. This could be due to the more diverse dynamics of hospital water distributions systems (Table 2). The hosts identified in the hospital settings consisted of three phyla, five classes and twelve genera, whereas the hosts isolated from potable water consisted of only two phyla, three classes and five genera. Two genera of Amoebozoa namely, *Vermamoeba* and *Acanthamoeba*, are frequently reported from both types of facilities as potential hosts. Available literature demonstrated that non-cyst-forming and ciliated protozoans can also be potential hosts for *L. pneumophila*. Most of the studies were designed specifically to explore the interactions between *L. pneumophila* - *Vermamoeba*/*Acanthamoeba*, and the diversity and the role of other possible protozoans were not investigated.

**Figure 1.** Overview of search methods and articles inclusion and exclusion criteria.


**Table 1.** Potential protozoan hosts of *Legionella pneumophila* isolated from and hospital and potable water systems.


**Table 1.** *Cont.*

#### *Pathogens* **2020**, *9*, 286


#### *Pathogens* **2020**, *9*, 286


**Table 1.** *Cont.*


*Pathogens* **2020**, *9*, 286

**Table 1.** *Cont.*


*Pathogens* **2020**, *9*, 286


**Table 1.** *Cont.*

reaction; ISO: International organization for standardization; MALDI-TOF MS: matrix assisted laser desorption ionization-time of flight mass spectrometry; qPCR: quantitative PCR; gu/L: genome unit/liter; LOQ: limit of quantification; LOD: limit of detection; EMA-qPCR: ethidium monoazide-qPCR; T-RFLP: terminal-restriction fragment length polymorphism; C/L: cells/liter; gc/mL: gene copy/milliliter; gc/L: gene copy/liter; CE/reaction: cell equivalent/reaction; CE/g: cell equivalent/gram; CE/L: cell equivalent/liter; \* CE/L: cyst equivalent/liter; gn/g: genome copy number/gram; gc/g: gene copy/gram; NGS: next generation sequencing; NEN: Nederlands normalisatie instituut; CE/cm2: cell equivalent/cm2; DVC-FISH: direct viable count combined with fluorescence in situ hybridization.

#### *Pathogens* **2020**, *9*, 286


**Table2.**Taxonomicdescriptionofpotentialprotozoanhosts.

In the studies identified, diverse physical and chemicals methods were used to disinfect the hospital and municipal potable water systems. Chlorination (<0.05–<4 mg/L) using different chlorine compounds was frequently reported as being used in both settings. Protozoans and *L. pneumophila* could still be isolated from both hospital and municipal potable water systems despite chlorination (<0.05–<4 mg/L), and/or ozonisation and thermal (<50–70 ◦C) disinfection protocols being in place. Importantly, several studies from hospital settings reported regular outbreaks of legionellosis. This represents a failure of existing disinfection protocols. The systematic literature review revealed that *L. pneumophila–Acanthamoeba*/*Vermamoeba* were extensively co-isolated from chlorinated and thermally treated water. This demonstrates the potential tolerance of *L. pneumophila* and protozoan hosts to survive under a wide range of disinfection conditions.
