Assessment of hygienic conditions of recreational facility restrooms: an integrated approach.

INTRODUCTION: Microbiological quality of recreational environments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility.
METHODS: Heterotrophic Plate Count (HPC) both at 22°C (psychrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR.
RESULTS: The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women's one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR).
CONCLUSIONS: Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and microbiological detection in an integrated approach could offer the advantage of greater sensitivity. ©2021 Pacini Editore SRL, Pisa, Italy.

Introduction

In recreational facilities, like swimming pools, people can get exposed to pathogens and this may constitute a public health problem; restrooms may represent a potential at-risk environment. The risk of acquiring infections in swimming pools is often associated with microbial contamination of water and inadequate disinfection. Additionally, direct contact with contaminated surfaces and inhalation of air are also potential routes of exposure to pathogens [1].

Legionella pneumophila (L. pneumophila) has been recovered from a wide range of artificial water systems including hot water supplies, cooling towers, whirlpools spa but it is also a common inhabitant of natural water [2].

Legionella spp. grows best in warm water, and many legionnaire disease outbreaks are associated with hot water systems [3, 4].

In public swimming pools, especially in the dressing room areas, L. pneumophila can spread through shower aerosols produced from contaminated water sources and aerosols can be inhaled [5].

Pseudomonas aeruginosa (P. aeruginosa) is also a common environmental bacterium that forms biofilms on wet surfaces [6]. It can also be found in groundwater and drinking water systems [7]. The bacterium is one of the most common causes of opportunistic human infections.

P. aeruginosa is responsible of many episodes of infections associated with attendance at swimming pools. Moreover, Legionella spp. and Pseudomonas spp. can also resist stressful environmental conditions, like water chlorination, because of their ability to enter into a viable but non-culturable (VBNC) state [8]. VBNC forms typically exhibit a low level of metabolic activity and fail to grow on standard medium, while they retain certain features of viable cells, such as cellular integrity and virulence [9]. Under favorable conditions they can resuscitate, recovering their cultivability and regaining pathogenic potential [10, 11].

Currently, there are several methods for Legionella spp. and Pseudomonas spp. detection and enumeration including: standard culture methods [12], PCR and real-time PCR [13, 14], also in its quantitative application [15]. Culture methods, using prescribed growth media and specific conditions, recover only culturable organisms [16]. Over the past ten years, molecular methods are used for the detection of airborne and waterborne microorganisms not growing on culture media.

Bioaerosols are among the less studied particles in the environment, due the lack of standardization in sampling procedures [17, 18]. Moreover, it has been showed that only a small proportion of the total bioaerosol burden is culturable [19].

Recommendations for the control and prevention of legionellosis [20] do not include air sampling. However, this kind of sampling has been used by other authors, in combination with water systems surveillance, for the detection of L. pneumophila [21] and P. aeruginosa [22]. Airborne L. pneumophila may be collected by agar impaction, filtration and liquid impingement. Agar impaction samplers should be used for short sampling times to avoid cell desiccation stress. However, in case of low bacterial concentration, false negative results can occur. To date, numerous liquid-and filter-based samplers have been used for detecting airborne Legionella [21]. Liquid-based sampling methods have been described as the best sampling methods to obtain culturable L. pneumophila [21, 23], while filtration sampling (gelatine filters) is adequate for capturing total cells. Indeed, collection with liquid impingement relieves the dehydration stress, although a major drawback with conventional impingers is the violent bubbling producing cell damage and reduction of microbial recovery. Some of these problems have been attenuated by swirling aerosol collectors, combining impingement and centrifugal motions to preserve cultivability [24].

The presence of L. pneumophila [25] and P. aeruginosa [13, 26] in recreational centers has been investigated by several authors by the examination of water through filtration membranes. In our knowledge, other sampling methods for the detection of the above-mentioned pathogens in sport-related environments, included restrooms, are lacking.

Therefore, the aim of this study was to evaluate if air microbiological monitoring, performed through a swirling aerosol collector (Coriolis), could give additional useful information about the hygienic conditions of recreational facility restrooms.

This pilot study aims to propose a new integrated approach, comprising standard microbiological methods and real-time PCR, for the detection of L. pneumophila and P. aeruginosa and the determination of the Heterotrophic Plate Count (HPC) in recreational facility restrooms, analyzing a complete panel of environmental matrices: water, surfaces and air.

This may offer the advantage of giving more representative data about microbial presence, including also bacterial species known to be transmitted through aerosol. Moreover, the concurrent use of molecular and microbiological detection could give greater sensitivity, especially with VBNC forms possibly originated from disinfection.

Methods

A total of 20 samples were collected from restrooms of one recreational swimming pool, between June and August 2018. This recreational swimming pool is in the Marche Region, Central Italy.

WATER SAMPLES

Each hot water sample, collected from sink taps and showers in men and women restrooms, was placed in 1 L sterile bottles, containing 10% sodium thiosulfate to neutralize any residual chlorine. Although all samples were collected by turning on the tap hot water, the temperature not exceeded 32°C. Samples were transported to the laboratory in a thermally insulated box and analysed immediately.

Legionella sampling and analysis was performed in accordance with Italian Guidelines [20] and the standard method ISO11731:2017 [27]. Briefly, 1 L of water was filtered through a membrane (0.2 μm, 47 mm diameter; Millipore, Billerica, MA, USA) of cellulose nitrate. Each membrane was put in a tube containing 5 ml of original water sample, shaken and then held at 50°C for 30 min. An aliquot of 0.5 ml was spread on Legionella CYE supplemented with Legionella BCYE growth supplement (Oxoid Ltd., Hampshire, UK) and incubated for 24 h at 37°C with 2.5% CO2 for 10 days. Suspected colonies were counted and then confirmed by real-time PCR, as described below.

Isolation and identification of P. aeruginosa was performed according to the standardized procedure UNI EN ISO 16266:2008 [12]. Briefly, 100 ml of each water sample was filtered with a cellulose ester membrane (0.45 μm porosity, 47 mm diameter; Millipore), which was then placed onto a Pseudomonas Agar with Pseudomonas CN Supplement (PACN) (Oxoid) plate. PACN plates were incubated at 35 ± 1°C for 44 ± 4 h before the counting of colonies. Blue/ green pyocyanin-producing colonies were counted as confirmed P. aeruginosa according to UNI EN ISO 16266:2008 [12]. Fluorescent non-pyocyanin-producing or reddish-brown colonies were recorded as presumptive P. aeruginosa and subjected to confirmation tests according to UNI EN ISO 16266:2008 [12].

The Heterotrophic Plate Count (HPC) at 22°C and 37°C was determined by the pour plate method, using standard Plate Count Agar (PCA, Oxoid) [28]. The plates were incubated at 37°C for 40-48 h and at 22°C for 64-72 h. The results were expressed in colony forming units (CFU)/ml.

AIR SAMPLES

Air contamination in recreational facility restrooms was assessed by a wet cyclone technology (Coriolis® μ Exonder, Borgo Ticino, NO, Italy). The Coriolis cyclone sampler was adjusted to sample 3,000 L of air (300 L/min for a period of 10 min). Airborne bacteria were collected in Coriolis® μ sterile cones filled with 15 ml phosphate buffered saline (PBS) + 0.005% Tween 80. HPC were conducted on PCA. 100 μl of sample were placed on the plates and incubated at 22 and 37°C. Next, the number of bacterial colonies was counted and recalculated as CFU per m3 (CFU/m3). The liquid material was filtered through 0.2 μm cellulose ester membranes of 47 mm diameter (Millipore). For isolation of Pseudomonas, filter was placed on Pseudomonas Agar Base with CN (Oxoid) and incubated at 35 ± 1°C for 44 ± 4 h. For isolation of Legionella, filter was placed on Legionella CYE Agar Base supplemented with Legionella BCYE growth supplement (Oxoid) and incubated for 24 h and 10 days, checking the plates at intervals of 2-4 days, at 2.5% CO2 at 37°C.

A second series of samples was analyzed in parallel by real-time PCR, as described below.

SWAB SAMPLES

Bacterial microbiota present on the surface of the shower head (inside and out) was collected using sterile non adsorbent cotton swabs rubbed on a 10 x 10 cm2 area, covering the entire surface by moving the swab back and forth across the surface horizontal and vertical strokes; then resuspended in 5 ml of physiological solution (0.9%, w/v, NaCl). For microbiological analyses, sample volumes of 0.1 mL were spread over the surface of PCA, PACN and Legionella CYE Agar Base supplemented with Legionella BCYE growth supplement. All plates were incubated for optimal temperature and time as per water analysis and after incubation the number of colonies was counted and was expressed as the number of CFU per cm2 (CFU/cm2).

PCR TESTING

Molecular detection in real-time PCR of P. aeruginosa and L. pneumophila was performed in water, air and swab samples. Sample analysis was performed according to Schiavano et al. [29] for P. aeruginosa and with the DI-Check Legionella pneumophila kit (Diatheva, Fano, Italy) for L. pneumophila.

Results

Concerning pathogen presence, both species were detected only in men restroom. P. aeruginosa was found in shower water and air samples by microbiological method and in shower swab surfaces by real-time PCR, while L. pneumophila was detected at a very low level in the external surface of shower heads with the real-time PCR, namely 4 and 7 genomic units (GU)/PCR, approximately corresponding to 160-280 GU in the sampled surfaces (Tab. I).

Tab. I.

Results of pathogen detection by microbiological and real-time PCR methods in samples from men restrooms.

Sample type		Microbiological methods		Real-time PCR
		P. aeruginosaUNI EN ISO 16266:2008	L. pneumophilaUNI EN ISO 11731:2017	P. aeruginosa	L. pneumophila
Water	Shower water	+	-	-	-
	Tap water	-	-	-	-
Air	Air	+	-	-	-
Surfaces	Shower head (inside)	-	-	+	-
	Shower head (out)	-	-	+	+*

* Values are: 4 and 7 GU/PCR, in the first and second sample, respectively.

The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women’s one (Tab. II). These values are in accordance with those reported by [30, 31].

Tab. II.

Results of HPC load in men and women restrooms.

Location	Sample type		HPC*
			22°C	37°C
Men restroom	Water	Shower water	< 10	> 100
		Tap water	< 10	< 10
	Air	Aerosol	> 500	> 300
	Surfaces	Shower head (inside)	n.c.	n.c.
		Shower head (out)	n.c.	n.c.
Women restroom	Water	Shower water	< 10	< 10
		Tap water	< 10	< 10
	Air	Aerosol	< 100	> 300
	Surfaces	Shower head (inside)	n.c.	n.c.
		Shower head (out)	n.c.	n.c.

n.c.: not counted. After incubation many colonies were confluent and this made counting impossible.

* Values are reported as CFU/ml for water samples and CFU/m3 for air samples.

Similarly, in air samples the HPC load of psychrophilic flora (22°C) was higher in men restroom respect to women’s one (Tab. II).

Discussion

Detection of microbial contamination in environmental matrices in recreational environments is important for safeguarding the state of hygiene and the health of pool users. The HPC at 22 and 37°C is a mandatory criterium to assess water potability, according to law provisions in Italy [32] and can be considered an indicator of hygiene. It is currently used to determine air quality in indoor environments, including recreational facilities [33].

In this study two different incubation temperatures were used to quantify the presence of mesophilic and psychrophilic species in water. According to the microbiological classification of air quality in non-industrial environments provided by the European Collaborative Action [34], a level of psychrophilic flora < 100 CFU/m3, which was found in women restrooms, corresponds to the category of “low contamination”, while an HPC load > 500, as that recorded in men restrooms, can be included in the “high contamination” category. Microbial pollution is a key element of indoor air pollution. It is caused by hundreds of species of bacteria and fungi, filamentous fungi (mold), growing indoors when sufficient moisture is available [35].

HPC are not considered an indicator of health risk. But, the Legionella presence in water appears to be in relationship with load and generic water quality parameters, i.e. HPC at 22°C and HPC at 37°C [36]. It is important to consider that the presence of these mesophilic and psychrophilic bacteria could be correlated with the colonization of water system by Legionella [37]. In our study, the water samples were collected from the hot water tap because numerous scientific evidences thought the hot water distribution system to be the most frequent source of cases or outbreaks of legionella in hotels, schools, sport facilities, offices and private residences [38].

In recreational facilities, the potential risk of infection with Legionella could be associated with the inhalation of aerosols containing the bacteria, in particular the aerosols created in the showers [39].

Although the limited sampling number does not allow a statistical analysis of significance about the association of high levels of HPC at 37°C and pathogen presence, which is out of the scope of this study, results confirmed previously reported data [25].

The lack of positive results for Legionella with the standard microbiological method could be due to the presence of VBNC cells [9] or bacteria contained in amoebae [10]. Moreover, PCR positive samples in absence of isolation of living bacteria could be ascribed to the presence of dead microorganisms, i.e. inactivated by disinfection procedures. Nevertheless, in water and air monitoring, filter-concentration before culture could have improved sensitivity, in contrast to real-time PCR, in which only a fraction of the extracted sample was PCR-amplified.

The integrated approach used in the present study takes advantage from the examination of different types of matrices, not limiting to water samples. Indeed, for some pathogens like L. pneumophila, aerosol inhalation is the main pathway of exposure, thus air sampling may offer useful information about its presence and give a better knowledge about the microbiological quality of such environments. Moreover, the application of two kinds of methodologies, culture-based and real-time PCR, increased detection sensitivity, especially for surface analysis.

Conclusions

Microbiological monitoring of water, air and surface quality for the presence of important species, like Legionella spp. and Pseudomonas spp., is useful and crucial in order to determine the potential exposure of swimming pool users. The use of different sampling methods gave integrated information that allowed to highlight the contamination by pathogens on different matrices.

The multipoint analysis approach used in this preliminary study, with the application of both culture and molecular methods, can increase the probability of a reliable detection.

In conclusion, an accurate environmental monitoring of restrooms in recreational pool facilities including air, along with the application of good hygienic practices, can be of main importance to prevent or reduce the exposition of pool users to microbiological risks.