A total of 108 water (swimming pool samples n = 41; hot tub samples n = 17) and swab samples (swimming pool samples n = 36; hot tub samples n = 14) were collected from eight swimming pools and three hot tubs. Twenty-three samples (21%) were positive for P. aeruginosa
, of which 16 were from hot tubs (70%); all samples from the residential hot tub were positive. Sixty-seven percent (2/3) of hot tubs and 63% (5/8) of swimming pools were positive at least once for P. aeruginosa. P. aeruginosa
-positive samples were much more likely to come from swabs than from water samples. The positive swab locations were diverse, including side-wall tiles, gutter drains, jets, and strainer baskets which may indicate that these are biofilm-rich locations. Table 1
shows the prevalence data.
Water analysis indicated low turbidity overall (0.1–0.6 NTUs), and a wide range of free (FC) and total chlorine (TC) levels (FC: 0.8 to >10.0 mg/L, average 8.46 mg/L; TC: 1.0 to >10.0 mg/L, average >10 mg/L). Swimming pool temperature ranged from 25.9 °C to 32.4 °C, and hot tub temperature 38.7 °C to 39.3 °C. Differences were noted between P. aeruginosa-positive and negative samples with respect to water temperature and free chlorine. Turbidity levels were very similar between the groups. Water quality data were not available for the residential hot tub (which comprised 63% of the P. aeruginosa positive hot tub samples).
All 23 isolates analyzed were resistant to at least one antimicrobial, and several were multidrug-resistant. Of the clinically relevant antibiotics included in the screening panel, there was resistance to amikacin (intermediate, 9%), aztreonam (22%), ceftriaxone (4% resistant, 30% intermediate), gentamicin (9%), imipenem (26%), meropenem (intermediate, 4%), ticarcillin/clavulanic acid (4%), tobramycin (intermediate, 9%), and trimethoprim/sulfamethoxazole (13% resistant, 17% intermediate). Table 2
shows minimum inhibitory concentrations (MIC) to these antibiotics. High levels of resistance were noted to eight additional antibiotics (e.g., ampicillin, nitrofurantoin, etc.
; See Table 3
), but these were not included in the MIC analysis as they are not clinically indicated for the treatment Pseudomonas aeruginosa
infections. Resistance is defined as an MIC result above the susceptibility upper bound, and intermediate resistance is defined as an MIC result above the susceptibility lower bound, but below the susceptibility upper bound.
The findings of this study indicate that P. aeruginosa
contamination is common in swimming pools and hot tubs, even where chlorine concentrations are well above recommended levels. Recommendations for adequate chlorine levels are 2 to 4 mg/L for swimming pools, and 3 to 5 mg/L for hot tubs, measured as free chlorine [23
]. According to NSPF recommendations, facilities should consider immediate closure when free chlorine levels fall below 1 mg/L, because contamination can quickly reach unsafe concentrations. With regard to hot tubs, prior research has demonstrated that even when hot tubs are drained, cleaned, and refilled, P. aeruginosa
may return with a level of 104
cells/mL within 24 to 48 hours when adequate chlorine levels are not maintained [15
]. In the current study, chlorine concentrations were higher than in past studies; and while P. aeruginosa
was routinely isolated, the regularly high chlorine levels found here likely reduced prevalence. This confirms the importance of not only adequate disinfectant levels, but also constant monitoring and adjustment to ensure chlorine levels never drop below recommended levels. It should be noted that the facilities in this study conducted hourly monitoring of pool and hot tub chlorine concentrations, except the residence, which performed weekly monitoring. The less frequent monitoring of the residential hot tub may have resulted in greater halogen fluctuations, and thus higher prevalence of P. aeruginosa
Consistent with previous research, a relationship between high chlorine levels, biofilm growth (as indicated by swab samples, which contain higher levels of biofilm-embedded bacteria, versus
water samples, which are more likely to represent planktonic bacteria), and the presence of P. aeruginosa
, was noted. LeChevallier et al.
(1988) have described several mechanisms leading to disinfectant resistance, of which attachment to surfaces was most important [24
]. They note that low-nutrient environments enhanced free chlorine resistance by two to three times. Thus, it is not surprising that this study indicated higher prevalence of P. aeruginosa
in pools/hot tubs with higher chlorine levels. There was also a relationship between higher water temperature and the presence of P. aeruginosa
. This finding is consistent with the thermo-tolerant nature of P. aeruginosa
There are several possible reasons why P. aeruginosa
was more likely to be isolated from hot tubs over swimming pools. Hot tub piping systems are complex and inaccessible for cleaning; this complexity may enhance biofilm formation [9
]. In addition, hot tub capacity is lower (per bather) than swimming pools (0.757 m3
/person for a hot tub versus
/person for swimming pools); this may act to concentrate microbial load present in the water [23
]. Finally, high pressure jets are an integral component of hot tubs, with bathers often using jets to enhance relaxation. Contact with jets in this manner may enhance sloughing of skin squames, further fouling hot tub piping systems.
The results of this study also indicated that environmental P. aeruginosa
has considerable levels of antibiotic resistance. Isolates demonstrated resistance to a wide range of clinically relevant antimicrobial agents, including antipseudomonal agents aztreonam (22%), gentamicin (9%), and imipenem (26%); and intermediate resistance to amikacin (9%), meropenem (4%), and tobramycin (9%). Resistance was also noted to alternate agents ceftriaxone (4% resistant, 30% intermediate), ticarcillin/clavulanic acid (4%), and trimethoprim/sulfamethoxazole (13% resistant, 17% intermediate). Although P. aeruginosa
is a model of antimicrobial resistance (due to a number of intrinsic factors), past research has found that the degree of resistance to antipseudomonal agents varies considerably. The current results are similar to past studies, which have found analogous prevalence levels of resistant P. aeruginosa
in both the hospital and community [4
The origins of multidrug-resistant strains in the swimming pool and hot tub environment are unknown, but may have developed through several mechanisms. Since P. aeruginosa
is intrinsically resistant, it is possible that the frequency with which we isolated resistant P. aeruginosa
is representative of natural background levels. Alternately, in some cases resistant P. aeruginosa
strains may ultimately be traced to a human source, either direct shedding from colonized humans or indirect transmission through the contamination in upstream aquatic reservoir. P. aeruginosa
is not a predominant component of normal human flora (colonization rates are low: up to 2% on the skin and to 24% in fecal matter) [4
]. However, during hospitalization, colonization rate may reach 50% [4
]. Also, immune suppressed individuals or those whom have recently undergone antimicrobial treatment may have higher colonization rates [4
]. In addition, in clinical settings, there is a strong relationship between the P. aeruginosa
strains which infect patients and those in the immediate patient environment. Rogues et al.
] pointed-out that carriage of P. aeruginosa
by patients was “both the source and the consequence of (tap) water colonization”. That is, a colonized or infected patient may lead to in-room and neighboring area contamination; and contaminated water, fomites, and aerosols may infect susceptible individuals [27
]. A similar phenomenon may occur in recreational water, particularly if frequented by colonized or infected individuals.
In some cases, there may be selective pressure for resistance by the presence of low-level antibiotic concentrations in either sweat secretions, which may enter recreational water through bather use, or from supply water. Prior research has demonstrated the presence of a range of antimicrobial agents in arm and axilla sweat secretions after oral or intravenous dosing [28
]. While isolated concentrations were found to be quite low, this is a possible mechanism for the development of resistant strains. Antibiotics have also been routinely isolated in water at various stages of processing, including in post-chlorination drinking water [29
]. Importantly, in some cases chlorinated water was found to select for more multidrug-resistant strains over non-chlorinated water [30
]. Given that the swimming pool and hot tub supply water in our study begins as municipal drinking water but then becomes highly chlorinated to reach disinfection levels, there may be enhanced selection for resistant strains.
While the levels of resistance among study isolates are similar to those previously reported, it is important to draw the distinction between a nosocomial setting and the non-clinical, non-outbreak, recreational setting of the current study. In clinical scenarios, there is constant selective pressure to enhance the proliferation of multidrug-resistant strains. Given that P. aeruginosa has both intrinsic resistance and a dynamic ability to develop resistance during the course of infection, a high frequency of resistance is now expected in hospitals. However, in the non-clinical environment, the absence of selective pressure may reduce antimicrobial resistance levels. The presence of resistance to front-line antipseudomonal drugs may have important clinical and prevention implications. Given how common swimming pool and hot tub use is, immune-suppressed or other high-risk individuals may wish to exercise caution before use, especially hot tub use. Exposure to resistant P. aeruginosa results in even greater risk for individuals with cystic fibrosis.
Study limitations include a relatively small sample size and the convenience design. Although only one residential hot tub was accessible for sampling in this study, there was a desire to broadly evaluate whether differences existed in the prevalence of P. aeruginosa and the degree of antimicrobial resistance. The residential hot tub owner did not experience previous skin diseases related to the hot tub and the hot tub did not undergo thorough maintenance and flush-fill cycles as did the public hot tubs, which were cleaned daily, drained completely once per week, and have fresh water introduced continually. Further study is required with expanded sample size to determine if the frequency of multidrug-resistant strains remains steady over time. Additional studies should focus primarily on hot tubs, where 53% of samples were positive. Also, isolates from three P. aeruginosa-positive samples were unable to be revived for antimicrobial resistance analysis.