Microorganism levels in spray from warm-water bidet toilet seats : factors affecting total viable and heterotrophic plate counts , and examination of the fl uctuations and origins of Pseudomonas aeruginosa

The objectives of this study were to conduct an appropriate microbial evaluation of warm-water bidet toilet seats. Health-related advantages and disadvantages have been associated with using warmwater bidet toilet seats, which are classified according to the tank type, including tanks equipped with reservoir water heaters and on-demand tankless systems equipped with an instantaneous water heater. However, related bacterial research is sparse. Here, we performed a long-term survey of the behavior of microorganisms (i.e., the total viable count (TVC), heterotrophic plate counts (HPCs), and Pseudomonas aeruginosa count) in a university campus. We also examined the differences between the tank and on-demand types, and the origins of P. aeruginosa. A low TVC ( 1/mL) in the spraywaters from both on-demand and tank-type warm-water bidet toilet seats showed low bacterial contamination, although there was an increase in HPC, i.e., growth of biofilms, inside in the warmwater bidet toilet seats. When P. aeruginosa was detected in spray water over an extended duration, the P. aeruginosa origin was considered as either from feces or tap water. Collectively our findings demonstrate that hygienic safety of warm-water bidet toilet seats is being maintained overall. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2017.137 om https://iwaponline.com/jwh/article-pdf/16/3/346/245948/jwh0160346.pdf 9 Toru Iyo (corresponding author) Department of Health Sciences, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan E-mail: iyot.ahs@kitasato-u.ac.jp Keiko Asakura Department of Environmental and Occupational Health, School of Medicine, Toho University, 5-21-16, Omori-Nishi, Ota-ku, Tokyo 143-8540, Japan Makiko Nakano Kazuyuki Omae Departments of Preventive Medicine and Public Health, Keio University School of Medicine, 35 Shinanomachi, Sinjuku-ku, Tokyo 160-8582, Japan


INTRODUCTION
Warm-water bidet toilet seats are equipped with a device that sprays warm water (spray water) on the external genitalia and anus after urination or defecation (Japan Monthly Web Magazine ).A consumer behavior survey conducted by the Japanese Cabinet Office showed that 79.1% of Japanese households have at least one such toilet seat, with every 100 households owning 110.9 units on average (effectively one per household) (Cabinet Office Government of Japan ).Broadly, two types of warm-water bidet toilet seats are available: the tank type and the on-demand type.With the tank type equipped with reservoir water heaters, the spray water is warmed to a suitable temperature in a tank with reservoir water heaters, whereas in the on-demand type tankless systems equipped with an instantaneous water heater, the water is warmed as needed inside a device with tankless, instantaneous water heaters (bidetsPLUS ) (Figure 1).Tanktype products are both cheaper and more common than on-demand types in Japan (Ministry of Economy Trade and Industry ).
Despite the reported health-related advantages (benefit to bowel movement, no clinical health risk for preterm birth and bacterial vaginosis, low incidence rates for bacterial vaginitis) of using warm water in bidets (Uchikawa et  Although Katano et al. () showed bacterial contamination (i.e., Pseudomonas aeruginosa and Escherichia coli isolated) of the spray water from the tank-type warm-water bidet toilet seats, research on both the amounts and the types of microorganisms present in the spray water is sparse.
Previously, we conducted a cross-sectional survey on the hygienic conditions of 127 warm-water bidet toilet seats in restrooms on a university campus, revealing relationships between the total viable count (TVC), heterotrophic plate count (HPC), and residual chlorine; the proportion of Pseudomonas aeruginosa in the spray water; and the composition of the microbiome in the spray water.We showed that the TVC, fecal indicator bacteria, and P. aeruginosa were present in the spray water at very low frequencies and concentrations, indicating that the overall hygienic safety was maintained (Iyo et al. ).
In this study, we conducted a long-term survey of the hygienic conditions of warm-water bidet toilet seats in a research building in a university campus (12 men's restrooms and one women's restroom), including restrooms in which P. aeruginosa was previously detected in the spray water (one men's restroom and one women's restroom).We thus empirically examined TVCs and HPCs, as well as P. aeruginosa behavior in the spray water; factors affecting these parameters (residual chlorine and differences between the tank and on-demand types); and the origins of P. aeruginosa from the viewpoint of preventing opportunistic infections due to P. aeruginosa.We also studied previously contaminated bidets for P. aeruginosa persistence, the age of the systems, variations over time, and the location of P. aeruginosa within the bidets.P. aeruginosa secretes a biopolymer that enables it to form biofilms and strong resistance to chlorine (Silva et al. ).The warm-water tanks in the bidets also promote biofilm formation and long-term survival of P. aeruginosa on the inner walls of these tanks.
Our aim was to conduct an appropriate microbial evaluation of the on-demand and tank-type warm-water bidet toilet seats in this study.

Warm-water bidet toilet seats
In a previous survey of 127 tank-type, warm-water bidet toilet seats, P. aeruginosa was detected in the spray water of only two (one men's and one women's restroom) in only one research building on a university campus (Iyo et al. ).The cause of the P. aeruginosa water contamination was not determined.Hence, focusing on the research building containing two warm-water bidet toilet seats where Pseudomonas aeruginosa was detected in the spray water, we surveyed warm-water bidet toilet seats in 12 men's and one women's restrooms in the research building (P.aeruginosa positive) on the same university campus, in this long-term field study.However, considering the difficulty for male researchers to collect the spray water from bidet toilet seats in women's restrooms, only one female bidet toilet seat in which P. aeruginosa was specifically detected in spray water was surveyed.The toilet seats were replaced with either tank-type or on-demand type seats in accordance with the study plan.Breakdown (disassembly) analysis was performed to detect P. aeruginosa, Escherichia coli, and enterococci in the piping of the toilet seats.

Analytical methods
The levels of residual chlorine (DPD method), TVCs (Com-pactDry ® TC; Nissui Pharmaceutical Co., Tokyo, Japan; growth at 37 C for 2 days), HPCs (R2A agar culture medium; Eiken Chemical Co., Tokyo, Japan; growth at 20 C for 7 days), P. aeruginosa (USEPA-Approved Defined Substrate Technologies Pseudalert ® and Quanti-Tray ® systems, IDEXX Laboratories Inc., Westbrook, ME, USA) and E. coli (USEPA-Approved Defined Substrate Technologies Colilert ® and Quanti-Tray ® systems, IDEXX Laboratories Inc., Westbrook, ME, USA) were measured in spray water and tap water (control), as described previously (Iyo et al. ).Samples of microbes in the internal piping and spray nozzles were collected by scraping the inside with a cotton swab or other similar tools.The collected samples were then inoculated into a nutrient broth (Eiken Chemical Co., Tokyo, Japan) and incubated at 37 C for 48 h.The incubated solution was then smeared on agar plates.The media used for smear culture were nalidixic acid cetrimide (NAC) agar medium (Eiken Chemical Co., Tokyo, Japan), sodium dodecyl sulfate magenta-GAL-X-GLUC agar medium (ELMIEX Limited, Tokyo, Japan), and Chromocult ® enterococci-agar (Merck KGaA, Damstadt, Germany) for P. aeruginosa, E. coli, and enterococci, respectively.The culture temperature and duration used with these media were based on the manufacturers' instructions.

PCR-based open-reading frame (ORF) typing classification of P. aeruginosa strains
To investigate the origins of P. aeruginosa, the culture medium for P. aeruginosa was diluted appropriately and subjected to isolation culture to obtain a few single colonies on NAC agar plates.Subsequently, DNA was extracted from the isolated colonies with the Cica-Geneus™ DNA Extraction Reagent (Kanto Chemical Co., Tokyo, Japan), and PCR-based ORF typing (POT) values (the combination of POT1 value and POT2 value) were measured using the CicaGeneus™ Pseudo POT (PCR-based ORF Typing) Kit (Kanto Chemical Co., Tokyo, Japan).Using the POT method with two sets of primer pairs, P. aeruginosa strains were classified by 10 ORFs from islets, 5 ORFs from genomic islands, and the metallo-beta-lactamases bla IMP and bla VIM (multidrugresistant Pseudomonas aeruginosa gene) (Suzuki et al.

Survey methods
Figure 2 shows the timelines for the surveys of the warmwater bidet toilet seats, which were conducted between 2012 and 2016 (Surveys 1-6).Survey 1: We followed up on the levels of P. aeruginosa in the spray water from a women's tank-type warm-water bidet toilet seat (TT-1f) and a men's tank-type warm-water bidet toilet seat (TT-1 m), wherein P. aeruginosa was detected in spray water from both in a previous survey.
Samples of spray water were collected in the morning.Survey 6: After confirming the P. aeruginosa levels and POT values in the spray and tank water of a women's tank-type warm-water bidet toilet seat (TT-1f) and in the spray water from a men's on-demand-type warm-water bidet toilet seat (OD-1 m), we examined the internal P. aeruginosa levels and POT values after dismantling these toilet seats.Subsequently, we prepared water piping from which tap water-derived P. aeruginosa was eliminated through microfiltration and then installed two on-demand-type warm-water bidet toilet seats (OD-1f and OD-1 m-2), using the piping.Spray water samples were collected from these seats and compared with two types of tap water as controls (Figure 3).Spray water and tap water samples were tested approximately once every 2 weeks for P. aeruginosa, E. coli, and enterococci.We also dismantled seats OD-1f and OD-1 m-2 thereafter to examine the presence and POT values of P. aeruginosa inside.

Statistical processing
While calculating the geometric means (GMs) and geometric standard deviations (GSDs) of residual chlorine, TVC, HPC, and P. aeruginosa levels, we also determined the detection limits.Statistical analysis of factors affecting the quality of spray water was conducted by performing analysis of variance (ANOVA).The quality of spray water from tank and on-demand types was compared by performing the Mann-Whitney U test and the non-parametric Kruskal-Wallis test.
Statistical analysis was performed using the Statistical Package for the Social Sciences, version 24 (IBM, Armonk, NY, USA).The significance level was set at P < 5%.

Fluctuations in P. aeruginosa levels
As indicated in Table 1, P. aeruginosa was detected in the same two tank-type warm-water bidet toilet seats (TT-1f and TT-1 m) that showed P. aeruginosa in a previous survey.In particular, P. aeruginosa was confirmed to have persisted for at least 2 years in the tank type warm-water showed P. aeruginosa in the spray water.One of the ondemand-type seats (OD-1 m) consistently showed low levels of P. aeruginosa in its spray water.As also indicated in Table 1, although one of the tank-type seats (TT-2 m-2) carried P. aeruginosa in its spray water before replacement, the bacterium disappeared after the seat was replaced with a new on-demand-type seat (OD-2 m).Thus, the only toilet seats that consistently showed P. aeruginosa were TT-1f and TT-1 m through OD-1 m (Survey 5).and TT-2 m-2).A weak negative correlation was also found between the residual chlorine and HPC levels (R: 0.491, R 2 : 0.241).

Influence of the collection time on the residual chlorine, TVC, and HPC levels
Figure 5 shows the daily and weekly variations of the residual chlorine, P. aeruginosa, and HPC levels.The only source of chlorine was the tap water, and the concentration of chlorine decreased due to heating and remaining stagnant for a long period.The graph shows an inverted U-shaped pattern in which the residual chlorine levels tended to be relatively low in the morning and peak around noon before dropping towards the evening.The HPC and P. aeruginosa levels, however, tended to be higher in the morning before decreasing in the evening.This result may reflect nocturnal growth of bacteria within the water tank, with subsequent washout and dilution of bacteria using spray water tank throughout the day.Alternatively, this result may reflect an inverse relationship between bacterial and chlorine levels.However, no weekly variations were observed.The difference in residual chlorine levels between TT-1m2 and TT-2 m-2 was likely due to the frequency of use.

Comparison between tank-type and on-demand-type toilet seats
After the tank-type toilet seats were replaced with ondemand-type seats, we compared the residual chlorine, TVC, and HPC levels in the spray water.Residual chlorine level increased more in the on-demand type than in the tank type, and HPCs decreased more in the on-demand type than in the tank type (P < 001; Figure 6).TVCs decreased more in the on-demand type than in the tank type, but the difference was not statistically significant (P ¼ 0.148; Figure 6).The on-demand-type of bidet toilet seat does not have a tank with reservoir water heaters, and the residual chlorine in tap water scarcely decreases because of the low degree of residual chlorine evaporation.
Consequently, the residual chlorine level in tap water was highly correlated with that in spray water (R: 0.963, R 2 : 0.926; Figure 7).

Origins of P. aeruginosa
We dismantled tank-type toilet seat TT-1f and on-demandtype toilet OD-1 m, both of which were consistently P. aeruginosa-positive, and analyzed the spray water (for both) and the tank water (for TT-1f).P. aeruginosa was detected near the filter in the piping and in the spray water in seat TT-1f, as well as in the piping within the nozzle and on the nozzle surface in seat OD-1 m (Figure 8).The P. aeruginosa POT values (i.e., POT1 and POT2) differed between TT-1f and OD-1 m.Table 2 shows the results of an experiment performed to determine the origins of P. aeruginosa, as described in Figure 3.
With seat OD-1f, only the tap water sample upstream of the microfilter (tap water 1) consistently showed high P. aeruginosa levels.In contrast, seat OD-1 m-2 occasionally showed P. aeruginosa in the spray water.The POT values in TT-1f were identical with those in OD-1f, but the POT values in OD-1 m and OD-1 m-2 were similar but not identical to each other.P. aeruginosa was not detected in the water from the washbasins of OD-1f and OD-1 m-2 in the restroom where these seats were installed (tap water 2).When OD-1f and OD-1 m-2 were dismantled for further investigation, P. aeruginosa was detected in the inlet of the microfilter for OD-1f and in the spray nozzle for OD-1 m-2 (Figure 8).The P. aeruginosa POT values differed between OD-1f and OD-1 m-2; in the case of the former, the POT values at the inlet of the microfilter were identical to those in tap water 1 (Table 2).With OD-1 m-2, P. aeruginosa was detected at the tip of the spray nozzle, but the POT values differed from those in the OD-1 m-2 spray water and tap water 1 (Figure 8 and Table 2).Thus, here, we reported a long-term survey of the hygienic conditions of warm-water bidet toilet seats in a university research building.Microorganism levels in spray water from warm-water bidet toilet seats showed that the overall hygienic safety was maintained.In a future study, an increased number of samples from warm-water bidet toilet seats is needed, especially for bidet toilet seats installed in female restrooms.In addition, to improve the hygienic safety of spray water from warm-water bidet toilet seats in their many different locations, self-cleaning spray nozzle mechanisms should be improved and structural changes should be made to prevent the growth of biofilms inside both warm-water tanks and pipes.

CONCLUSIONS
We performed a long-term survey of the behavior of microorganisms within warm-water bidet toilet seats.The TVCs were 1/mL in spray water samples from both on-demand and tank-type warm-water bidet toilet seats.The HPCs in the spray water were 4 log 10 in the tank type and 3 log 10 in the on-demand type.However, the residual chlorine and HPC levels in the spray water were only weakly correlated.When Pseudomonas aeruginosa was detected in spray water over an extended duration, the bacterial origin was likely feces or tap water.The POT values of presumably tap water-derived P. aeruginosa were uniform, whereas those of presumably feces-derived P. aeruginosa varied.
The isolated P. aeruginosa strain was unlikely to possess enzymes conferring multidrug resistance.Microorganism levels in spray water from warm-water bidet toilet seats showed that the overall hygienic safety was maintained in these facilities.
al. ; Asakura et al. ; Kiuchi et al. ), there are also healthrelated disadvantages (aggravation of vaginal microflora, correlation for an itch on the anus) associated with the use of these toilet seats (Ogino et al. ; Tsunoda et al. ).

Figure 1 |
Figure 1 | Schematic diagram of two types of warm-water bidet toilet seats.

Survey 2 :
To eliminate the effects of ageing of the warmwater bidet toilet seats, two toilet seats (TT-1 m and TT-2 m) were replaced with new ones (TT-1 m-2 and TT-2 m-2).After approximately half a year of acclimation to stabilize the state of the biofilm in the toilet seats, spray water was surveyed in each toilet seat.Data from seats TT-1 m-TT-7 m were used as control for those from seats TT-1 m-2-TT-2 m-2.Spray water samples were collected in the morning.Survey 3: We investigated daily (five times a day at 9:00, 12:00, 15:00, 18:00, and 21:00) and weekly variations (five weekdays from Monday to Friday) of residual chlorine, HPC, and P. aeruginosa levels without disinfecting the nozzle surface.Survey 4: We examined the fluctuations in P. aeruginosa levels in spray water from seven men's tank-type warmwater bidet toilet seats (TT-1 m-2, TT-2 m-2, TT-3 m, TT-4 m, TT-5 m, TT-6 m, and TT-7 m) without disinfecting the nozzle surface.Survey 5: In total, 12 men's tank-type warm-water bidet toilet seats (TT-1 m-2, TT-2 m-2, TT-3 m, TT-4 m, TT-5 m, TT-6 m, TT-7 m, TT-8 m, TT-9 m, TT-10 m, TT-11 m, and TT-12 m) were replaced with new, on-demand-type seats (OD-1 m, OD-2 m, OD-3 m, OD-4 m, OD-5 m, OD-6 m, OD-7 m, OD-8 m, OD-9 m, OD-10 m, OD-11 m, and OD-12 m).We surveyed the quality of spray water once a month between August and December.Spray water samples were collected in the morning.When comparing the spray water quality between the toilet seat types, we used data from November for both the tank-type and on-demandtype toilet seats because data for the tank type were collected in November.

Figure 2 |
Figure2| Experimental timeline showing when samples of spray water were collected from a women's tank-type warm-water bidet toilet seat (TT-1f) and 12 men's tank-type warm-water bidet toilet seats (TT-1 m-2, TT-2 m-2, TT-3 m, TT-4 m, TT-5 m, TT-6 m, and TT-7 m).P. aeruginosa levels were determined from all the samples before (Survey 1), after seat replacement from tank type to tank type (Survey 2) or after seat replacement from tank type to on-demand type (Survey 5).In addition, residual chlorine and heterotrophic plate counts were measured during Survey 3 and P. aeruginosa levels and PCR-based ORF typing values were measured in the spray and tank water during Survey 6.

Fluctuations
Figure 4(a)).As shown in Figure 4(a), the TVC was nearly undetectable in the spray water from seats TT-1 m-2 and TT-2 m-2, and even when TVC was detected, the value did not exceed 10 CFU/mL.In addition, a weak negative correlation was observed between the residual chlorine and HPC levels (correlation coefficient [R]: 0.457, coefficient of determination [R 2 ]: 0.209).As shown in Figure 4(b), the TVCs were 1/mL in the spray water from the on-demand-type warm-water bidet toilet seats.When the TVCs were detected in the spray water, they tended to decrease as the residual chlorine levels increased.Most toilet seats (9 out of 11; 82%) that showed a TVC exceeding 1 CFU/mL were the on-demandtype seats (OD-1 m and OD-2 m) that replaced the previously P. aeruginosa-positive tank-type seats (TT-1 m-2

Figure 3 |
Figure 3 | Schematic representation of the experimental equipment used for investi- gating the origins of P. aeruginosa.

Figure 5 |
Figure 5 | Temporal variations in the residual chlorine, HPC, and P. aeruginosa levels in spray water from tank-type toilet seats (a) TT-1 m-2 and (b) TT-2 m-2.

Figure 6 |
Figure 6 | Comparison of the residual chlorine, TVC, and HPC levels in spray water between tank-type and on-demand-type toilet seats.

Figure 7 |
Figure 7 | Correlation between residual chlorine levels in tap water and spray water in on-demand-type toilet seats.

Figure 8 |
Figure 8 | Localization and POT values of P. aeruginosa in tank-type (TT-1f) and on-demand-type (OD-1f, OD-1 m, and OD-1 m-2) toilet seats in the origin test, using on-demand-type toilet seats.

Table 2 |
Genotyping and molecular epidemiological analysis of Pseudomonas aeruginosa from warm-water bidet toilet seats (Survey 6) MPN, most probable number; POT, PCR-based open reading frame typing.a Coliforms-positive.b E. coli-positive.