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Article

Comparative Risk Assessment of Legionella spp. Colonization in Water Distribution Systems Across Hotels, Passenger Ships, and Healthcare Facilities During the COVID-19 Era

by
Antonios Papadakis
1,2,*,
Eleftherios Koufakis
3,
Elias Ath Chaidoutis
4,
Dimosthenis Chochlakis
1,5 and
Anna Psaroulaki
1,5
1
Department of Clinical Microbiology and Microbial Pathogenesis, School of Medicine, University of Crete, Voutes—Staurakia, 71110 Heraklion, Greece
2
Public Health Authority of the Region of Crete, 71201 Heraklion, Greece
3
Civil Protection of the Region of Crete, 71201 Heraklion, Greece
4
First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece
5
Regional Laboratory of Public Health, School of Medicine, 71110 Heraklion, Greece
*
Author to whom correspondence should be addressed.
Water 2025, 17(14), 2149; https://doi.org/10.3390/w17142149
Submission received: 10 June 2025 / Revised: 16 July 2025 / Accepted: 17 July 2025 / Published: 19 July 2025
(This article belongs to the Special Issue Legionella: A Key Organism in Water Management)

Abstract

The colonization of Legionella spp. in engineered water systems constitutes a major public health threat. In this study, a six-year environmental surveillance (2020–2025) of Legionella colonization in five different types of facilities in Crete, Greece is presented, including hotels, passenger ships, primary healthcare facilities, public hospitals, and private clinics. A total of 1081 water samples were collected and analyzed, and the overall positivity was calculated using culture-based methods. Only 16.46% of the samples exceeded the regulatory limit (>103 CFU/L) in the total sample, with 44.59% overall Legionella positivity. Colonization by facility category showed the highest rates in primary healthcare facilities with 85.96%, followed by public hospitals (46.36%), passenger ships with 36.93%, hotels with 38.08%, and finally private clinics (21.42%). The association of environmental risk factors with Legionella positivity revealed a strong effect at hot water temperatures < 50 °C (RR = 2.05) and free chlorine residuals < 0.2 mg/L (RR = 2.22) (p < 0.0001). Serotyping analysis revealed the overall dominance of Serogroups 2–15 of L. pneumophila; nevertheless, Serogroup 1 was particularly prevalent in hospitals, passenger ships, and hotels. Based on these findings, the requirement for continuous environmental monitoring and risk management plans with preventive thermochemical controls tailored to each facility is highlighted. Finally, operational disruptions, such as those experienced during the COVID-19 pandemic, especially in primary care facilities and marine systems, require special attention.

1. Introduction

Water distribution systems are important parts of public infrastructure because they provide clean drinking water and help stop the spread of waterborne diseases [1,2,3]. Nonetheless, under certain physicochemical and operational conditions, these engineered systems can unintentionally become breeding grounds for opportunistic pathogens, such as Legionella spp. (especially L. pneumophila), which are gram-negative bacteria that cause Legionnaires’ disease, a severe form of atypical pneumonia that causes significant illness and death, especially in older people and people with weakened immune systems [4,5,6,7,8,9]. According to the ECDC Legionnaires’ Disease Annual Epidemiological Report for 2021, 10,723 cases were reported in 29 countries, of which 10,004 (93%) were confirmed [10]. The rate of notifications per 100,000 people increased to 2.4, which is higher than any other year. Italy, France, Spain, and Germany together made up 75% of recorded cases, even though their populations only made up about 50% of the EU/EEA population. Of the 8054 cases with a known outcome, 704 ended in death [10].
Legionella loves warm water and is quite good at living in constructed systems with stagnant water, inadequate treatment, and the right temperature ranges [11]. Biofilms and host protozoa allow them to live longer and spread faster. Outbreaks often happen in high-risk places, such as hospitals, hotels, and ships, because their plumbing is complicated, they do not use water all the time, and they have different ways of keeping things clean [12,13,14,15,16,17].
Studies have shown that water systems in buildings, such as hotels, hospitals, and ships, are quite likely to be colonized by Legionella, making them potent places for Legionella colonization [18,19,20,21,22,23,24,25,26]. For instance, Alexandropoulou et al. found that L. pneumophila was quite common in Greek healthcare facilities, and temperature and system design affected its spread [19]. Naher also found that hotel hot water systems are high-risk places, pointing to poor temperature management and the lack of water safety plans (WSPs) as two of the main reasons [20]. Papadakis et al. examined hotel water systems across the island of Crete [21]. Between 2000 and 2019, approximately 63% of the hotels inspected, after a case notification of Legionnaires’ disease, were colonized with Legionella spp. [21]. The study also highlighted the important things that need to be managed in water distribution systems to remain clean and disinfected. One of these was the successful implementation of WSPs to improve water supply and sanitation systems of hotels.
Long-term building closures and less water use during the COVID-19 pandemic made it easy for Legionella to spread [22,23,27,28]. Nationwide lockdowns were implemented in March 2020, November 2020, and January 2021; in 2022, the measures were relaxed. These periods were characterized by limited access to healthcare and disorientation from the normal maintenance of public infrastructure, including water systems. It is therefore likely that the measures affected water use and stagnation patterns, particularly in the health and hospitality sectors [29]. Rhoads and Hammes pointed out that microbial growth increased during the lockdown-related standstill, demonstrating the importance of flushing measures [30]. During the pandemic, Kunz et al. observed that U.S. hotels and motels were less likely to follow Legionella control strategies, which made the risk of outbreaks even higher [31].
Public health authorities are increasingly calling for integrated control measures, such as WSPs that use the principles of Hazard Analysis and Critical Control Points (HACCP), to help reduce the risk of legionellosis in complex healthcare and hospitality systems [32,33] and that have demonstrated effectiveness in reducing Legionella risk in complex healthcare and hospitality systems [34,35]. Disinfection strategies involving the application of agents, such as monochloramine, chlorine dioxide, and hydrogen peroxide, have demonstrated efficacy in reducing Legionella colonization, particularly when embedded within comprehensive monitoring and maintenance frameworks [36,37]. However, the dynamics of Legionella colonization remain highly context dependent, necessitating localized risk assessments that consider building design, usage patterns, and endemic microbiological profiles.
To our knowledge, this is the first environmental surveillance study in Greece to systematically assess Legionella colonization trends across multiple high-risk facility types—including hospitals, outpatient clinics, hotels, and passenger ships—during and after the COVID-19 pandemic.
The present study aims to evaluate and compare the colonization levels Legionella spp. across various facilities on the island of Crete from 2020 to 2025. This study aims to identify facility-specific risk factors and assess the impact of pandemic-related disruptions on water system hygiene, thereby informing future prevention strategies and water safety policy implementation in complex aquatic environments.

2. Materials and Methods

2.1. Study Design and Sample Collection

This multiparametric environmental monitoring study was conducted from March 2020 to March 2025, covering the entire territory of the Region of Crete, Greece. The study was conducted by the Public Health Authority of the Region of Crete in collaboration with the Regional Public Health Laboratory and the Department of Clinical Microbiology and Microbial Pathogenesis.
Sampling included five categories of facilities: (1) hotels—accommodation facilities; (2) passenger ships; and three different health care facilities, including (3) primary care units, (4) public hospitals and (5) private clinics (Table 1). The five facility categories were selected based on international guidelines from the European Centre for Disease Prevention and Control (ECDC) and the World Health Organization (WHO), which classify healthcare facilities (hospitals, clinics, outpatient centers) and hospitality facilities (hotels, ships) as high-risk environments for Legionella propagation. In total, 41 facilities were included in the study, which were distributed as follows: 13 hotel units, 10 public hospitals, 6 primary care units, 6 passenger ships and 6 private clinics. The basic selection of facilities was carried out (a) due to their inclusion in regular public health inspections and regional surveillance programs, (b) after a reporting of a Travel-Associated Legionnaires’ Disease (TALD) case related to travel and identified through the European Legionnaires’ Disease Surveillance Network (ELDSNet) (especially in hotels), or (c) as a random sampling carried out by public and environmental health officials of the Region of Crete for the needs of active surveillance following standardized procedures. Samples employed by the 41 different facilities were studied to ensure a wide spatial and typological distribution.
The systematic sample collection included 1081 water samples from multiple points in the water distribution network for each facility. Table 1 lists the sampling points, which include municipal pipe inlets, water storage tanks, hot water boilers and heaters; terminal outlets (showers, faucets, and taps in patient or visitor rooms), swimming pools and hot tubs, where applicable, and outlets proximal or peripheral to the facility’s primary water source. The strategy intentionally targeted high-risk nodes within the facility’s plumbing systems, with an emphasis on aerosol-generating outlets as well as known outlets based on temperature profiles or user reports associated with colonization. Their grouping based on sampling characteristics was direct vs. indirect flow paths; cold vs. hot water; and proximal (close) vs. distal (far) outlet locations.
The samples were obtained following international and European standards, namely ISO 5667-1:2020 and ISO 5667-1:2023, as well as the European Technical Guidelines for the Prevention, Control and Investigation of Infections Caused by Legionella spp. (ECDC) [38].
Briefly, the following sample collection procedure was followed. A sterile 1-L container containing 20 mg of sodium thiosulfate was used to neutralize the presence of disinfectant residues. The samples were stored at a temperature of 5 ± 3 °C, under shaded conditions and transported to the regional microbiology laboratory for analysis within 24 h under controlled conditions. The physicochemical parameters were measured directly on-site using calibrated, portable devices. Measurements included the following: free residual chlorine concentration (mg/L), water temperature (°C) after 2 min of rinsing, pH levels, turbidity where applied, sampling point type (direct vs. indirect; hot vs. cold; near vs. far), facility metadata (disinfection method, capacity, water source, and presence or absence of a documented water safety plan (WSP)).
These environmental parameters were measured on-site at the time of sampling using calibrated portable instruments. These variables were categorized according to internationally accepted limits for the analysis. Specifically, hot water < 50 °C, free chlorine < 0.2 mg/L, and cold water > 20 °C were used.
The relationship between environmental exposure and temporal variation in Legionella colonization was studied by monitoring three key physicochemical parameters for each sample: hot water and cold water temperature (°C) and free residual chlorine (mg/L). To identify associations, annual noncompliance was measured and compared with positivity rates. These variables were also included in the multivariate logistic regression model to assess their independent predictive value during the surveillance period.

2.2. Microbiological Analysis

Microbiological testing for the detection and quantification of Legionella spp. from water samples was performed by culture according to the International Standard method ISO 11731:2017 [39].
Briefly, water samples were concentrated by filtration and resuspended in distilled deionized water. A volume of the suspension (200 μL) was spread on (BCYE), Buffered Charcoal Yeast extract without l-cysteine (BCY), and Glycine Vancomycin Polymyxin Cycloheximide (GVPC) agar (Biomérieux, Craponne, France). For the Petri dishes (culture plates): (a) directly after filtration; (b) after incubation at 50 °C for 30 min; and (c) after the addition of an acid buffer (0.2 mol/L solution of HCL, pH 2.2). The detection limit of the procedure was 50 CFU/L. The inoculated plates were incubated for up to 10 days at 36 ± 1 °C under 2.5% CO2 atmosphere with increased humidity. Suspected colonies were randomly selected for subculture on BCY, BCYE, and GVPC agar.
A matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Microflex LT) (Bruker Daltonics, Leipzig, Germany) equipped with a micro-SCOUT ion source was used to identify individual Legionella colonies against its microbial database (v 3.1.2.0). Spectra were recorded using the flex Control software version 3.4 with the manufacturer’s default optimization parameters (Bruker Daltonics, Leipzig, Germany). For each spectrum, 240 laser shots were collected and analyzed (6 × 40 laser shots from 120 different positions of the target spot). All identifications were evaluated according to the manufacturer’s scoring scheme.
Acid and heat pretreatment steps were employed to reduce background flora and enhance Legionella recovery. The detection limit of the method was 50 CFU/L, allowing for reliable quantification within the regulatory action range. Confirmed colonies were further serotyped to distinguish L. pneumophila serogroups (especially SG1 vs. SG2–15) and identify non-pneumophila species, providing key epidemiological insights into pathogen virulence and potential disease risk.

2.3. Risk Assessment and Statistical Analysis

To better calculate the risk, two different approaches were used. The first one was in accordance with the recommendations of the European Legionnaires’ disease Surveillance Network (ELDSNet), at which the microbiological results of the water samples were analytically and statistically analyzed according to the number of Legionella bacteria in the water sample, which could represent a particular risk to human health. An insignificant risk was noted at ≤103 CFU/L, medium risk at >103 CFU/L but <104 CFU/L, and high risk at ≥104 CFU/L.
All statistical analyses were conducted using the IBM SPSS Statistics Version 30 statistical package, the Epi Info 2000 build 7.2.7.0 (Centers for Disease Control and Prevention, Atlanta, GA, USA), and the MedCalc relative risk calculator statistical software free online version [40,41,42]. Descriptive statistics included frequencies, proportions, and Wilson 95% confidence intervals. Relative Risks (RR) with 95% confidence intervals were calculated for key noncompliant conditions, and independent predictors of positivity were identified using logistic regression. Categorical risk variables from water distribution systems and faculty characteristics associated with Legionellae-positive test results were analyzed. Linear regression was used to explore trends and quantify associations between Legionella concentrations and physicochemical parameters. The results were considered statistically significant when the p value was <0.05 and highly significant when the p value was p < 0.0001. The proportion of Legionella-positive samples was calculated for each water source and outlet type. To estimate the precision of these proportions, 95% confidence intervals (CIs) were calculated using the Wilson score interval method, which is appropriate for binomial data, particularly when sample sizes are small or proportions are near 0 or 1. All CIs were computed using a standard normal approximation with z = 1.96 z = 1.96 z = 1.96.

2.4. Multivariate Logistic Regression

A multivariate logistic regression model was developed to evaluate the independent predictors of Legionella spp. positivity (culture based, ≥50 CFU/L). Specifically, (a) the presence of Legionella spp. ≥ 50 CFU/L was used as the dependent variable and (b) facility type, sample type (hot vs. cold), sampling year, hot water temperature (<50 °C vs. ≥50 °C) and free residual chlorine (<0.2 mg/L vs. ≥0.2 mg/L) were used as independent variables.
Private clinics from the facility categories under study, the year 2020, cold water from the sample type, temperatures ≥ 50 °C and ≥0.2 mg/L chlorine residual were selected as reference. The selection criteria were public health standards or the lowest observed risk. Among the facility types, private clinics had the lowest observed Legionella positivity rate, with zero highly positive samples. Next, 2020 was used as a reference value due to the lowest annual positivity, since it marks the beginning of the pandemic. In the sample types, the choice of cold water is necessary as it is generally associated with a lower risk of Legionella compared to hot water. Finally, the limits for hot water temperature (≥50 °C) and free residual chlorine (≥0.2 mg/L) are standards for WHO and ECDC and thus were selected as protective reference levels. Odds ratios (ORs), 95% confidence intervals (CIs) and p-values were reported for all predictors. SPSS v30.0 and the MedCalc Relative Risk Calculator were used to conduct the analysis.

3. Results

3.1. Legionella Positivity and Temporal Trends

Of the 1081 samples collected from all five facilities, 482 (44.59%) tested positive for Legionella spp., with concentration levels varying from 50 to more than 10,000 CFU/L. The positive results are listed in Table 2, divided into three groups according to low (<103 CFU/L), medium (≥103 to <104 CFU/L), and high concentration levels (≥104 CFU/L). An enhanced positivity rate of samples was found for the primary healthcare units, with 85.96% testing positive and a significant 20.17% placed in the high-risk category (≥104 CFU/L). In contrast, public hospitals showed a lower overall positivity rate of 46.36%, with only 1.81% of the samples exceeding the higher threshold. Private clinics had the lowest overall positivity rate (21.42%). No samples had more than 104 CFU/L, and only 6.25% of them were in the medium-risk group. Next, samples from passenger ships showed a positivity rate of 36.93%, with 6.25% of them exceeding the 104 CFU/L threshold. Finally, hotels had a moderate positivity rate of 38.08%, and most of the positive samples were below the 103 CFU/L threshold. Nevertheless, 10.04% were in the medium-risk range and 4.60% were over 104 CFU/L. According to the European Drinking Water Directive (EU) 2020/2184, Legionella spp. concentrations equal to or surpassing 103 CFU/L require immediate remedial actions [43]. Based on this standard, 178 samples (16.46%) from all types of facilities exceeded this legal limit.
Figure 1 shows the positivity rate on the legal limit threshold of ≥103 CFU/L as a function of facility types. The primary healthcare units showed an enhanced level of contamination with 36.83% of total samples above the threshold, while a very moderate positivity rate was shown for public hospitals (17.94%), and hotels (14.64%). Passenger ships and private clinics showed a much lower level of contamination (8.52%), and a minimal positivity rate (6.25%).
The positivity rate for the surveillance of Legionella spp. for a six-year period between 2020 and 2025 was studied to elucidate further. The temporal analysis shown in Figure 2 shows a profound variation in the positivity rates of Legionella over the predetermined period. In 2020, the positivity rate was 14.75%, referring to a period before the onset of COVID-19 pandemic. An abrupt increase was observed in 2021, with 58.82% positivity, followed by a sharp reduction in 2022 at 14.71%. In the following years, 2023 and 2024, the positivity rates decreased gradually to 11.76% and 15.15%, respectively, with the standardization of the operating regulations. Nevertheless, in 2025, an almost 4-fold increase in positivity rate at 57.53% is observed, which is comparable to the post-lockdown period. Furthermore, the sampling distribution was consistent over time (Table S1), which presents the annual distribution of samples for the monitoring period from 2020 to 2025 to account for possible biased unequal sampling for each year in each facility type. In addition, to explore potential justifications for these temporal variations and recognize the environmental factors that contributed to the positive trends, the percentage of samples with thermal and chemical non-compliance was tracked annually and is further analyzed in Section 3.3 (Figure 3).
Next, analysis of the sample types revealed a higher presence of Legionella species in hot water systems than in cold water systems. Table 3 summarizes the Legionella-positive samples by sample type and collection point. Overall, 252 samples tested positive out of 481 hot water samples in total, which is ascribed to 52.28%, whereas 230 samples tested positive for Legionella, at 47.42% among 600 cold water samples in total. For hot water samples, positivity rates found a negligible difference between direct outlets and indirect outlets, 50.71% and 50%, respectively. Nevertheless, distal sampling points demonstrated a lower positivity rate at 48% than the proximal sampling points at 34.37%, potentially reflecting thermal decay and sediment buildup at terminal points. For cold water samples, negligible differences were observed between direct and indirect outlets, while far-away points showed a slightly lower positivity rate of 23.80% than the close points at 28%.

3.2. Serogroup Distribution and Facility Colonization Risks

To determine the distribution of Legionella species and serogroups for all facilities, 482 Legionella-positive samples were serotyped. As shown in Figure 4 and listed in Table 4, the majority of the isolates (65.35%) were Serogroups 2–15 of L. pneumophila. Serogroup 1 of L. pneumophila, often associated with clinical diseases, accounted for 36.09% of the positive samples. Only 2.70% were associated with species other than pneumophila. Serogroup 3 (11.38%) was the dominant serogroup among Serogroups 2–15 of L. pneumophila. Next, Serogroup 8 accounted for 7.49%, Serogroup 6 covered 5.00%, Serogroup 2 represented 1.57%, Serogroup 7 represented 0.65%, Serogroup 9 accounted for 0.37%, and Serogroup 4 formed 0.19%. Public hospitals recorded the highest number of positive samples (204·42.32%), with the distribution of serogroups favoring SG2–15 (54.41%) over SG1 (46.56%). Out of 98 samples from primary care units (20.33%), SG2–15 was the predominant type, constituting 81.63%. This suggests that the strains are spreading in the environment and may exhibit reduced pathogenicity. Next, passenger ships (13.49%) and hotels (18.88%) exhibited similar serogroup patterns, mainly consisting of SG2–15. In contrast, private clinics represented only 4.98% of all positive samples, with 70.83% of these classified as SG2–15. Non-pneumophila species were not detected. L. pneumophila Serogroup 1 was mainly found in public hospitals (95 isolates; 46.56% of positives in hospitals), followed by passenger ships (36.92%) and hotels (38.59%).

3.3. Temporal Evolution, Positivity Trends and Post-COVID Risk Profile

The temporal trend of Legionella positivity and environmental non-compliances was determined from 2020 to 2025. Figure 3 presents the annual Legionella positivity in relation to the percentage of environmental samples that did not comply with the parametric values set by public health authorities and were consistently monitored at each sampling point. More specifically, the hot water non-compliance (<50 °C), low free chlorine levels (<0.2 mg/L), and cold water > 20 °C, present peaks with highly increased positivity rates with a pronounced peak in 2021, and a subsequent resurgence in 2025 for selected facility types such as hotels.
Furthermore, Figure 3 shows declines in the water quality and Legionella proliferation between 2020 and 2025. It is noted that 2020 is the initial pandemic year. A relatively low positivity rate, 26.42%, was observed in 2020. Nonetheless, the chlorine levels (<0.2 mg/L) were 31.58% of the samples and extensive hot water temperatures were below the 50 °C safety standard. In 2021, there was an extensive increase in Legionella positivity, with its value rising to 59.17%. This rise coincides with a significant increase in water quality violations since chlorination and temperature parameters showed chlorine insufficiency (<0.2 mg/L) and temperature noncompliance in hot 75.53% and cold 63.04% samples respectively.
On the other hand, in 2022 and 2023, the positivity rates exhibited a modest level compared to the year 2021, with values dropping to 34.97% and 39.22%, respectively. The samples obtained in these years showed better compliance with chlorine levels, mainly due to strict regulations during COVID-19. Nevertheless, in 2024, non-compliances and violations re-emerged, with 45.56% of samples not meeting the chlorine criteria and 51.48% of cold-water samples exceeding 20 °C. In 2025, there was a significant improvement with only 18.75% of samples above the limit; however, the remaining parameters remained suboptimal, with the overall positivity increasing again to 59.23%.

Hotels: Post-COVID Risk Profile

A focused analysis of hotel establishments (n = 9) from 2020 to 2025 and their comparison with the pre-pandemic era demonstrates the impact of the pandemic on water system hygiene. Table S2 lists the rates of Legionella positivity in hotels for the period 2020–2025. In 2020, Legionella positivity in hotels was at a level of 26.42%, slightly lower than the national average of positivity in hotels of 38.08% for the period 2000–2019 reported in our previous study [21]. However, a rapid increase in positivity was observed during and after the COVID-19 pandemic, reaching 59.17% in 2021 and again 59.23% in 2025, values that far exceed both the 2020 baseline and the national average of positivity in hotels for the period 2000–2019. It is noted that the 2021 peak coincided with the widespread reopening of facilities after a prolonged period of inactivity. Partial improvement was noted in 2022 and 2023, with positivity falling to 34.97% and 39.22% respectively, but levels increased again in 2024, with values of 41.19% reaching a second peak in 2025.
In Addition, Table S2 lists the environmental risk factors monitored alongside positivity. In 2021, 48.45% of hotel samples with Legionella detections had free chlorine levels below 0.2 mg/L, 75.53% of hot water samples were below 50 °C and 32.50% of cold water samples exceeded 20 °C. These non-compliances align with periods of higher colonization and correspond to a more than two-fold increased risk for positivity (Table 5). Thermal and chemical instability remained problematic throughout 2025, while there were compliances for cold water (10.52% > 20 °C).
A comparative analysis of concentration zones (Table S3) also showed a shift from high risk (>104 CFU/L) to more persistent low- and medium-risk colonization in the post-COVID years.

3.4. Environmental Risk Factors and Relative Risk Analysis

Environmental and operational risk factors are then presented in Table 5. Hot water temperature below 50 °C emerged as the most critical with a relative risk (RR) of 2.05 and odds ratio (OR) of 6.54 for Legionella positivity (p < 0.0001). Also striking was the effect of hot water below 35 °C, which showed an RR of 3.58 and an OR of 4.74, reflecting a significantly increased colonization potential in hypothermophilic conditions. Free residual chlorine concentrations below 0.2 mg/L were also significantly associated with colonization (RR = 2.22, p < 0.0001), demonstrating the importance of continuous chemical disinfection (Table 5).
A weak but statistically significant association (RR = 2.53, p = 0.04) was found with increased turbidity levels, which is likely due to the support provided by sediment and biofilm for microbial growth (Table 5). As expected, no significant association was found between Legionella positivity and cold-water temperatures above 25 °C (RR = 1.12, p = 0.1), sampling locations in remote areas (RR = 1.11, p = 0.22) or direct water outlets (RR = 0.96, p = 0.29).
Statistical analysis can highlight which parameter is critical in Legionella proliferation. Table 5 shows the relative risk (RR) of Legionella positivity in relation to three important physicochemical factors of non-compliance: cold water temperature > 20 °C, free residual chlorine < 0.2 mg/L and hot water temperature < 50 °C. An RR greater than 1 means that the specified condition makes contamination more likely. The strongest association is seen with non-compliance with the hot water rules (RR > 2), which is due to low chlorine levels. A weak, non-significant association is shown for high values of non-compliance for cold water temperatures.
Linear regression supported these results, showing a strong association between non-compliance with hot water regulations and Legionella detection (β = 3578.0, p < 0.0001), with a moderate explanatory power (r2 = 0.07). There was a weak association between lack of free chlorine (β = 366.5, p = 0.0897) and cold water temperature that had no statistically significant predictive value (β = 245.4, p = 0.2056). Spearman’s rank correlation supported these patterns by showing a strong unidirectional association between Legionella detection and non-compliance with hot water regulations (ρ = 0.42, p < 0.0001).
Systemic vulnerabilities can be identified by the correlation of physicochemical parameters with non-compliances by facility type (Table S4). Of all samples, 61.06% was found to be non-compliant for hot water temperatures (<50 °C). Passenger ships and private clinics showed the highest rate with 92.31% and 65.78%, respectively. In advance, passenger ships found to present low residual chlorine (<0.2 mg/L) with 63.33% and primary healthcare units 42.19%, 36.34% of all samples displayed low residual chlorine (<0.2 mg/L), while non-conformities on cold water temperature violations (>20 °C) were found in 87.76% of cases, mostly 89.57% of cases attributed to hotel samples. Finally, pH values were observed with no deviations, implying that chemical buffering systems remained within acceptable ranges across all facilities.
Table S2 presents the temporal trends in the prevalence of risk factors among Legionella-positive samples. In 2021 and 2025, years characterized by increased positivity, there were simultaneous peaks of non-compliance with thermal and chlorinated requirements. Linear regression confirmed the predictive power of suboptimal hot water temperatures for colonization (β = 3578.0, p < 0.0001; r2 = 0.07), while chlorine deficiency showed a weaker association (β = 366.5, p = 0.0897). Cold water temperature did not show significant predictive value (p = 0.2056).
Finally, the risk assessment of positive samples based on CFU/L specifications (Figure 5) showed that high-risk samples (≥104 CFU/L) made up a larger percentage of samples in primary care units (20.17%) and on passenger ships (6.25%). Private clinics had no high-concentration samples, whereas public hospitals and hotels had significantly fewer (1.81% and 0.57%, respectively).

3.5. Multivariable Analysis Results

A logistic regression model was performed to evaluate the independent predictors of Legionella positivity. The adjusted odds ratios (AOR), 95% confidence intervals (CI), and p-values for all included variables are summarized in Table S5. Private clinics, 2020 reference year, water temperatures < 50 °C, chlorine levels < 0.2 mg/L and cold water sample type were used as reference points for comparison. The probability of positivity was significantly higher in primary care units (AOR = 17.50), followed by public hospitals (AOR = 3.21), hotels (AOR = 2.34), and passenger ships (AOR = 2.09) compared to the reference category. Also, the sampling years 2021 and 2025 are associated with significantly increased odds of positivity relative to the reference year 2020. Hot water temperatures < 50 °C (AOR = 2.88) and free chlorine levels < 0.2 mg/L (AOR = 1.92) were associated with an increased risk of positivity. Finally, the sample type (hot vs. cold) had a marginal but significant effect (AOR = 1.35, p = 0.045).

4. Discussion

This 6-year, large-scale study analyzed the presence of Legionella spp. colonies in water distribution systems from 5 different high-risk facilities in Crete, Greece covering the period before, during, and after the COVID-19 pandemic. The findings highlight specific environmental and critical operational factors that influence Legionella proliferation and its footprint in water distribution systems.

4.1. Facility-Specific Colonization Patterns

In this study, an overall positivity rate of 44.59% for all Legionella species colonizing the water supply systems of the facilities is revealed. This is in agreement with the corresponding surveillance studies across Europe. However, our analysis shows that there is a different risk profile per facility. The highest positivity rate of 85.96% is presented in primary healthcare units, with a parallel presence of a high percentage of samples ≥ 104 CFU/L at 20.17%. In similar findings, Whiley et al. previously showed that decentralized healthcare settings are more prone to Legionella contamination [44].
Public hospitals showed a positivity rate at lower levels of 46.36%, although this was still significant. In these facilities, the positive fact is that only 1.81% of the samples exceeded 104 CFU/L. However, the presence of widespread levels of Serogroup 1 of L. pneumophila, with 46.56% of the positive samples, comprises a crucial risk factor, as Serogroup 1 has been recognized for its virulence and its involvement in epidemics of hospital infections. These results are consistent with the study by Buse et al., which examined large water tanks in large plumbing systems where persistent Legionella colonization occurred in healthcare settings [45]. Public health authorities should promote the adoption of such innovations, with the aim of saving energy, maintaining water infrastructure, and ensuring the hygiene and integrity of the water supply [45,46,47].
On passenger ships, the overall positivity was 36.93% but showed a high concentration of samples ≥ 104 CFU/L at 6.25% and the highest frequency of non-compliance with hot water temperature at 92.31%. Previous studies by Leoni et al., highlighted the high risk of Legionella colonization on ships due to the lack of ample space for redesigning systems and the difficulty in maintaining a continuous water flow [48]. This poses a unique challenge for microbial control on passenger ships; therefore, coordination between central and local authorities is essential to ensure preparedness and develop targeted risk assessments and contingency plans and to inform national strategies for water safety in maritime infrastructure [49,50].
Hotels exhibited a moderate positivity rate of 38.08%. Nevertheless, further analysis revealed fluctuations in Legionella infection risk over time between 2020 and 2025 (Figure 4, Supplementary Table S2). Positivity rates experienced two peaks, one in 2021 at 59.17% and the second in 2025 at 59.23%, with exceedances well above the baseline level in hotel facilities, before the pandemic.
A notable difference was in the non-compliance with the high cold water temperature, which was found at 59.55%. A significant percentage of samples, at 13.29%, presented a medium load rate (within 103–104 CFU/L) with a small presence above 104 CFU/L, which predisposes to low-level colonization.
The positivity rates from hotels, where visitors were reported as TALD cases in ELDSNet (n = 13) and the visitor’s room was known (n = 6), were found to be 55% over the total 40 samples, with the majority belonging to L. pneumophila Serogroup 2 at 22.73% [51]. The risk in accommodation facilities, such as guest rooms, bungalows, and hotel units, has been attributed before to complex plumbing infrastructure, intermittent water use and features, such as saunas and hot tubs [52,53].
The association of peak values between these temporal and functional factors is supported by the observed increase in environmental non-compliance in the same years, particularly for subcritical hot water temperatures and inadequate chlorine levels. As shown in Figure 3, the years with higher positivity rates corresponded to an increased relative risk. This relationship was further confirmed by multivariate logistic regression (Section 3.5), which identified hot water temperatures < 50 °C (AOR: 2.88) and low chlorine levels < 0.2 mg/L (AOR: 1.92) as significant independent predictors of Legionella colonization. These findings reinforce the fact that the temporal evolution of positivity reflects real changes in exposure conditions and not sampling bias.
In addition, both facility type and sampling year are significant predictors of positivity, confirming the interaction between facility characteristics and changes in their operation during the pandemic (Table S5).
In contrast to the phenomena of increased positivity rate and non-compliance observed in the remaining health and non-health facilities that we mentioned, private clinics presented the lowest rates in all parameters. It is noted that for the entire reporting period, the analysis included zero high-load samples. The image reflected for private clinics is that they have an advantage due to newer infrastructure and plumbing networks, but they also operate under strict accreditation standards with high compliance with cleaning and disinfection protocols. Nevertheless, there is a possibility of colonization even in low-risk environments, as shown by the presence of Serogroups 2–15 of L. pneumophila in 70.83% of positive samples.

4.2. Serogroup Distribution and Environmental Risk Indicators

Seroepidemiology in all facilities revealed the predominant presence of Serogroups 2–15 of L. pneumophila, with 63.83% of the positive samples. This is fully in line with the rules of environmental surveillance, where it is common to find the presence of Serogroups 2–15 in environmental isolates, compared to SG1, which appears less frequently and mainly in clinical cases [18]. Nevertheless, in the present study, a high prevalence of SG1 was observed in public hospitals, hotels and passenger ships, which reinforces the requirement for conducting seroepidemiological studies and the necessity of regular serogroup typing during environmental surveillance.
The study of physicochemical parameters identified suboptimal hot water temperature (<50 °C) and low free residual chlorine (<0.2 mg/L) as statistically significant risk factors. Multivariate logistic regression confirmed hot water non-compliance as the strongest independent predictor (AOR: 2.88; β = 3578.0; p < 0.0001), followed by inadequate chlorine levels (AOR: 1.92). The relative risk of colonization was more than doubled (RR = 2.05) in association with hot water systems maintained below 50 °C, while even higher values (RR = 3.58) were observed when temperatures fell below 35 °C. According to the WHO and the CDC, thermal control is a cornerstone of Legionella prevention and is confirmed by the findings of this study [54,55].
In contrast, the analysis of physicochemical parameters, in all samples, shows that cold water temperatures > 20 °C, remote sampling locations, and direct versus indirect exits were not statistically significant factors for the prediction of colonization. While these factors may influence risk under certain conditions, they appear secondary to thermal and chemical controls in this dataset.

4.3. Temporal Trends and the COVID-19 Effect

Temporal trend studies provided interesting information about the impact of operational disruptions on water safety. In 2021, which is the year after the first lockdown related to the COVID-19 pandemic, the highest positivity rate was recorded. According to international studies, water stagnation, maintenance suspension and generally reduced use of facilities in the reference facilities contributed to a trend of proliferation of all microbes, including those of Legionella spp. [56,57].
A smaller secondary increase in positivity was noted in 2025, particularly in association with certain non-compliant water parameters (e.g., hot water temperatures < 50 °C and free chlorine levels < 0.2 mg/L). These findings are consistent with the study by Kunz et al., which reported a lack of Legionella transmission control in US hotels during the pandemic [31]. The present study suggests a similar vulnerability in hotels in Crete during 2021 and 2025.

4.4. Strengths and Limitations

This study was conducted in an analysis based on a large sample size, a longitudinal 624 design and a typological diversity. The geographical restriction to a single island region 625, as well as the plate counting method for Legionella detection constitute limitations of the 626 study. Future studies should incorporate molecular diagnostics, such as qPCR and se- 627 sequence-based typing (SBT), to enhance detection sensitivity and monitor strain evolution.

4.5. Public Health Implications

In summary, this study highlights the need to develop and implement risk management strategies, differentiated and adapted to each facility. There is a requirement for targeted surveillance in both public hospitals and primary care settings, taking into account vulnerable patient populations. Seasonal risk assessments and planning of a water safety plan on board are a priority for passenger ships. Hotel operators should emphasize thermal controls even during off-season periods with low occupancy and ensure continuity in water use. Finally, in private clinics, the appearance of low risk may be a positive sign but should not be overlooked as minimal colonization can present serious risks for immunocompromised patients and vulnerable groups.

5. Conclusions

Legionella colonization in hotel water systems has been shown to increase during the COVID-19 pandemic and has remained elevated post-COVID. We concluded that infection rates, bacterial loads and environmental risk factors vary significantly per facility, with primary health care units being the most vulnerable ones. This highlights the complexity of water system design, operational practices, and public health outcomes. Moreover, our findings indicate systemic deficiencies in water safety monitoring, thermal regulation and disinfection practices, particularly in smaller outpatient clinics. Our findings also demonstrate the need for the implementation of maritime-specific protocols because passenger ships have traditionally not been included in water safety frameworks. The implication of inappropriate temperatures and hypochlorination in the presence of Legionella in water distribution systems urges the proper implementation of ESGLI guidelines.
Facilities should apply individualized risk assessment approaches, one of which is the implementation of integrated WSPs. WSPs should emphasize regular monitoring, automated thermal control and remedial disinfection protocols.
Future research should incorporate molecular diagnostics, strain typing, and digital monitoring tools to improve detection sensitivity and enable early warning systems. Enhancing the resilience of engineered water systems against Legionella and other microbial threats will depend on the integration of microbiological surveillance, engineering design, and adaptive governance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w17142149/s1, Table S1. Number of water samples collected from 2020 to 2025 per facility, Table S2. Year-by-Year Legionella Positivity and Water Quality Indicators in Hotel Water Systems (2020–2025), Table S3. Comparative Distribution of Legionella-Positive Samples by CFU/L Concentration Band, Table S4. Frequency and percentage of non-compliance with physicochemical parameters by facility type and year of monitoring (2020–2025), Table S5. Multivariable Logistic Regression Results for Legionella Positivity.

Author Contributions

For conceptualization, methodology, and validation A.P. (Antonios Papadakis), E.K., D.C. and A.P. (Anna Psaroulaki) are responsible; formal analysis, investigation, and data curation, was conducted by A.P. (Antonios Papadakis), E.K., E.A.C., D.C. and A.P. (Anna Psaroulaki); writing—original draft preparation, A.P. (Antonios Papadakis) and E.K.; writing—review and editing, A.P. (Antonios Papadakis) and A.P. (Anna Psaroulaki); supervision, and project administration A.P. (Antonios Papadakis), D.C. and A.P. (Anna Psaroulaki) All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to thank all the environmental health inspectors of the Local Public Health Authorities of Crete Island who collected the environmental samples during all these years.

Conflicts of Interest

The authors have declared no conflicts of interest.

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Figure 1. Legionella positivity rates (>50 CFU/L) by facility type, with 95% Wilson confidence intervals. The highest rates were observed in primary healthcare units and public hospitals. Error bars represent 95% CIs around the observed proportions.
Figure 1. Legionella positivity rates (>50 CFU/L) by facility type, with 95% Wilson confidence intervals. The highest rates were observed in primary healthcare units and public hospitals. Error bars represent 95% CIs around the observed proportions.
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Figure 2. Annual positivity rate of Legionella spp. in environmental water samples from various facilities in Crete (2020–2025). Black bars represent the total number of samples collected per year, and grey bars indicate the number of samples testing positive for Legionella spp. The red line shows the annual positivity rate (%) along with 95% confidence intervals (Clopper–Pearson method), providing a measure of statistical uncertainty.
Figure 2. Annual positivity rate of Legionella spp. in environmental water samples from various facilities in Crete (2020–2025). Black bars represent the total number of samples collected per year, and grey bars indicate the number of samples testing positive for Legionella spp. The red line shows the annual positivity rate (%) along with 95% confidence intervals (Clopper–Pearson method), providing a measure of statistical uncertainty.
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Figure 3. Temporal comparison of Legionella positivity and physicochemical non-compliance (2020–2025). Yearly percentages of positive samples (grey), free chlorine levels < 0.2 mg/L (green), cold water > 20 °C (blue), and hot water < 50 °C (red) across all facilities.
Figure 3. Temporal comparison of Legionella positivity and physicochemical non-compliance (2020–2025). Yearly percentages of positive samples (grey), free chlorine levels < 0.2 mg/L (green), cold water > 20 °C (blue), and hot water < 50 °C (red) across all facilities.
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Figure 4. Distribution of Legionella serogroups in positive samples (n = 482) with 95% confidence intervals.
Figure 4. Distribution of Legionella serogroups in positive samples (n = 482) with 95% confidence intervals.
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Figure 5. Risk classification of Legionella spp. positive samples by facility type. Risk categories defined according to ELDSNet as low (<103 CFU/L), medium (103–<104 CFU/L), and high (≥104 CFU/L). Primary health care units exhibit the highest proportion of high-risk samples (20.17%), followed by passenger ships (6.25%) and hotels (4.60%).
Figure 5. Risk classification of Legionella spp. positive samples by facility type. Risk categories defined according to ELDSNet as low (<103 CFU/L), medium (103–<104 CFU/L), and high (≥104 CFU/L). Primary health care units exhibit the highest proportion of high-risk samples (20.17%), followed by passenger ships (6.25%) and hotels (4.60%).
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Table 1. Distribution of water samples by facility and sampling site characteristics (2020–2025).
Table 1. Distribution of water samples by facility and sampling site characteristics (2020–2025).
FacilityTotal SamplesClose PointFar PointDirectIndirectCold WaterHot Water
Public Hospitals440 (40.70%)27 (6.1%)39 (8.9%)244 (55.5%)178 (40.5%)245 (55.7%)196 (44.5%)
Hotels239 (22.11%)15 (6.3%)21 (8.8%)128 (53.6%)101 (42.2%)133 (55.6%)106 (44.4%)
Passenger Ships176 (16.28%)11 (6.3%)16 (9.1%)102 (58.0%)74 (42.0%)98 (55.7%)78 (44.3%)
Primary Health Care Units114 (10.55%)7 (6.1%)10 (8.9%)66 (57.9%)48 (42.1%)63 (55.6%)51 (44.4%)
Private Clinics112 (10.36%)6 (5.9%)10 (8.9%)60 (53.6%)47 (42.0%)62 (55.7%)50 (44.3%)
Total1081 (100%)66 (6.1%)96 (8.9%)600 (55.5%)436 (40.3%)600 (55.5%)481 (44.5%)
Table 2. Legionella positivity by facility type (2020–2025).
Table 2. Legionella positivity by facility type (2020–2025).
FacilityNumber of SamplesTotal Positive Samples (>50 CFU/L)95% CI (Wilson)<103 CFU/L≥103 and <104 CFU/L≥104 CFU/L
Public Hospitals (tertiary-level care)440204 (46.36%)41.76–51.03%125 (28.40%)71 (16.13%)8 (1.81%)
Private Clinics11224 (21.42%)14.84–29.91%17 (15.17%)7 (6.25%)0
Primary Health Care11498 (85.96%)78.41–91.17%56 (49.12%)19 (16.66%)23 (20.17%)
Hotels23991 (38.08%)32.15–44.37%55 (23.01%)24 (10.04%)11 (4.60%)
Passenger ship17665 (36.93%)30.15–44.27%50 (28.40%)4 (2.27%)11 (6.25%)
Total1081482 (44.59%) 303 (28.03%)125 (11.56%)53 (4.90%)
Table 3. Legionella-positive samples by sample type and collection point.
Table 3. Legionella-positive samples by sample type and collection point.
Sample TypeNumber of Samples (n)Positive (%)95% CI
Hot water (total)48152.28%47.7–56.8%
└─ Direct outlet25850.71%44.6–56.7%
└─ Indirect outlet20650.00%43.1–56.9%
└─ Far away point5048.00%34.4–61.9%
└─ Close point3234.37%19.6–52.2%
Cold water (total)60047.72%43.7–51.8%
└─ Direct outlet34239.18%34.0–44.5%
└─ Indirect outlet23036.08%29.8–42.8%
└─ Far away point4623.80%13.1–38.2%
└─ Close point3428.00%14.4–45.9%
Table 4. Distribution of Legionella serogroups across facility types, with 95% confidence intervals. Percentages of L. pneumophila Serogroup 1, Serogroups 2–15, and other Legionella species were calculated based on the total number of positive samples in each facility type. All proportions are presented with their corresponding 95% confidence intervals (Wilson score method).
Table 4. Distribution of Legionella serogroups across facility types, with 95% confidence intervals. Percentages of L. pneumophila Serogroup 1, Serogroups 2–15, and other Legionella species were calculated based on the total number of positive samples in each facility type. All proportions are presented with their corresponding 95% confidence intervals (Wilson score method).
FacilityTotal Positive Samples (n, %, 95% CI)L. pneumophila SG1 (n, %, 95% CI)SG2–15 (n, %, 95% CI)Other Legionella spp. (n, %, 95% CI)
Hotels91 (18.88%, 15.6–22.6%)27 (29.60%, 20.7–40.6%)66 (72.53%, 62.4–81.1%)6 (6.59%, 3.0–13.6%)
Passenger ships65 (13.49%, 10.6–16.9%)24 (36.92%, 26.0–49.4%)41 (63.08%, 50.6–74.0%)0 (0.00%, 0.0–5.52%)
Primary Health Care98 (20.33%, 17.0–24.0%)21 (21.43%, 14.2–30.9%)80 (81.63%, 73.0–88.1%)0 (0.00%, 0.0–3.69%)
Private Clinics24 (4.98%, 3.4–7.1%)7 (29.17%, 14.9–49.2%)17 (70.83%, 50.8–85.1%)0 (0.00%, 0.0–14.25%)
Public Hospitals204 (42.32%, 38.8–45.9%)95 (46.57%, 39.8–53.5%)111 (54.41%, 47.4–61.0%)7 (3.43%, 1.7–6.9%)
Total482 (44.59%, 40.6–48.6%)174 (36.09%, 31.8–40.5%)315 (65.35%, 61.0–69.5%)13 (2.70%, 1.6–4.6%)
Table 5. Risk factors for Legionella colonization per physicochemical parameters and water sampling sites.
Table 5. Risk factors for Legionella colonization per physicochemical parameters and water sampling sites.
Risk FactorOdds Ratio (Cross Product)Odds Ratio (MLE)Risk Ratio (RR)Risk Difference (RD%)p-Value
Free residual chlorine < 0.2 mg/L2.22132.21752.217518.6545<0.0001
Cold water temperature > 20 °C1.85891.85691.517913.54370.01
Hot water temperature < 50 °C6.53946.50732.05441.5504<0.0001
Hot water temperature < 35 °C4.73654.72023.579222.3196<0.0001
Turbidity (non-compliant)2.56622.56392.53131.35030.04
Direct water sample (vs. outlet)0.93410.93420.9610−1.65620.29
Sampling at distal (far) outlet point1.35341.35041.11246.89150.22
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MDPI and ACS Style

Papadakis, A.; Koufakis, E.; Chaidoutis, E.A.; Chochlakis, D.; Psaroulaki, A. Comparative Risk Assessment of Legionella spp. Colonization in Water Distribution Systems Across Hotels, Passenger Ships, and Healthcare Facilities During the COVID-19 Era. Water 2025, 17, 2149. https://doi.org/10.3390/w17142149

AMA Style

Papadakis A, Koufakis E, Chaidoutis EA, Chochlakis D, Psaroulaki A. Comparative Risk Assessment of Legionella spp. Colonization in Water Distribution Systems Across Hotels, Passenger Ships, and Healthcare Facilities During the COVID-19 Era. Water. 2025; 17(14):2149. https://doi.org/10.3390/w17142149

Chicago/Turabian Style

Papadakis, Antonios, Eleftherios Koufakis, Elias Ath Chaidoutis, Dimosthenis Chochlakis, and Anna Psaroulaki. 2025. "Comparative Risk Assessment of Legionella spp. Colonization in Water Distribution Systems Across Hotels, Passenger Ships, and Healthcare Facilities During the COVID-19 Era" Water 17, no. 14: 2149. https://doi.org/10.3390/w17142149

APA Style

Papadakis, A., Koufakis, E., Chaidoutis, E. A., Chochlakis, D., & Psaroulaki, A. (2025). Comparative Risk Assessment of Legionella spp. Colonization in Water Distribution Systems Across Hotels, Passenger Ships, and Healthcare Facilities During the COVID-19 Era. Water, 17(14), 2149. https://doi.org/10.3390/w17142149

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