3.1. Characteristics of swimming pools
This study was conducted during summer months, when the swimming pools (Sp) are mostly used by people. The table-S1 shows the daily average number of bathers, the precaution methods before entering the swimming pools, source of water for the swimming pools, water exchange, disinfection method of the eight selected swimming pools. Each swimming pool depends on one or two sources of water, including (well water, tanker water, and water distribution system). In addition, pool water exchange was taken place according to the filtration system (table-S1), and several precautionary actions were taken to prevent the spread of waterborne diseases and algal growth. Chlorine was continuously added to the swimming pools through manual or automatic system within different time intervals (table-S1). According to WHO (2006) recommendation both filtration and proper application of chlorine disinfectant are necessary to control the threat of viruses and bacteria in swimming pool water [6].
Due to their affordability and practicality, chlorine disinfectant is the most effective and commonly used disinfectant in municipal water supply networks [36]. Pool water quality from public and semi-public pools should be monitored to ensure the water safety of pool users and protect them from any negative health impacts that caused by chemical and microbiological contaminants of water [1, 21, 22]. Previous study by Skibinski et al. (2016) confirmed that water re-circulation system and continuous chlorine injection are extremely required to mitigate microbial activity in swimming pools and to provide safety for the pool users [ 37]
3.2. Physicochemical analysis and their correlation with bacterial content
During summer time (August to September), the swimming pool water’s pH, electrical conductivity, and temperature are more suitable for bacterial growth and survival. In the current study, the results of physicochemical analysis of water samples collected from eight public swimming pools were showed that temperature ranged between (25.81±1.16 to 28.69±1.03°C), pH ranged from (7.02±0.24 to 7.46±0.31), and water conductivity (334±30 to 487±29 µS cm− 1) (table-1). Moreover, results of present study showed great fluctuation in the mean values of free chlorine (0.12±0.05 to 1.38±1.24 mg L− 1) (Table 1).
According to WHO (2006) the degree of hypochlorous acid dissociation into H + and OCl– (hypochlorite ion) depends on pH and on temperatures [38]. Results of physicochemical water analysis, the range of water temperature in accordance with the range of recorded temperature in swimming pools of Egypt [39], and the pH values in accordance with the WHO permissible limits (6.5–8.5) [25].
Table 1
Shows the number of bacterial isolates (n) and mean values of physicochemical parameters (Mean± S.D) collected from eight public swimming pools in Sulaymaniyah city.
Swimming pools
|
Physicochemical parameters
|
No. of bacterial isolates
|
Percentage
%
|
Temperature (°C)
|
Conductivity
µS cm− 1
|
pH values
|
Free chlorine (mg L− 1) ppm
|
SP1
|
26.00±0.55
|
462±67
|
7.23±0.17
|
0.17±0.09
|
11
|
18.64
|
SP2
|
25.81±1.16
|
487±29
|
7.14±0.25
|
0.45±0.47
|
4
|
6.80
|
SP3
|
26.89±0.87
|
388±23
|
7.16±0.23
|
0.18±0.13
|
8
|
13.55
|
SP4
|
27.33±0.86
|
334±30
|
7.02±0.24
|
0.12±0.05
|
9
|
15.25
|
SP5
|
27.64±1.00
|
491±24
|
7.23±0.15
|
1.38±1.24
|
6
|
10.16
|
SP6
|
28.25±1.25
|
367±26
|
7.46±0.31
|
0.23±0.10
|
10
|
16.94
|
SP7
|
28.69±1.03
|
469±29
|
7.26±0.22
|
0.28±0.16
|
6
|
10.16
|
SP8
|
27.95±1.19
|
448±36
|
7.20±0.18
|
0.42±0.25
|
5
|
8.50
|
Person correlation was used to determine the strength of correlation between number of bacteria isolates with physicochemical parameters of water samples, as well as, Correlation among physical–chemical parameters themselves (water temperature, free chlorine, pH). Results of statistical analysis showed that bacterial isolates and pH values with weak positive (r = 0.24) non-linear correlation with pH values, moderate negative non-linear correlation (r= -0.45) with free chlorine, and no sensible correlation was found between water temperature and bacterial isolates. In consistence to this finding Luo et al., (2018) emphasized that increasing in free chlorine significantly reduced the bacterial population in the medium [40]. Moreover, weak correlation (r = 0.09) was detected between (pH values and free chlorine), and (r = 0.11) between (water temperature and free chlorine); moderate positive non-linear correlation was found between water temperature and the pH values with r = 0.44 (Table 2). Weak correlation between free chlorine and both temperature and pH can be explained by the degree of hypochlorous acid dissociation into H+ and OCl– (hypochlorite ion) depends on both pH and temperatures [38].
Table 2
Person correlation between bacterial isolates and physiochemical parameters (water temperature, pH, and free chlorine), and person correlation between physiochemical parameters.
Statistical analysis
|
Bacterial isolates
vs.
Water temperature
|
Bacterial isolates
vs.
pH values
|
Bacterial isolates
vs.
Free chlorine
|
(r) Pearson correlation
|
-0.07
|
0.24
|
-0.45
|
p-values
|
0.86
|
0.56
|
0.26
|
Level of significant p < 0.05
|
ns
|
ns
|
ns
|
Statistical analysis
|
Water temperature
vs.
pH values
|
Water temperature
vs.
Free chlorine
|
pH values
vs.
Free chlorine
|
(r) Pearson correlation
|
0.44
|
0.11
|
0.09
|
p-values
|
0.27
|
0.79
|
0.83
|
Level of significant p < 0.05
|
ns
|
ns
|
ns
|
3.3. Bacteriological evaluation
Microbiological analyses of pool water samples that were collected weekly along two successive months (August to September 2021). Water analysis mainly focuses on detection of fecal coliform as a microbial indicator for water contamination [26]. According to the findings of the microbiological analysis, all water samples had negative MPN results, which suggests that no coliform group had been found in any of the tested swimming pools. This indicates that non-lactose fermenter bacteria are dominant over lactose fermenter coliform group in swimming pools, which will ultimately raise the potential risk on swimmers that are frequently visiting the swimming pools. In support to this explanation, previous study by Cabral (2010) confirmed that chlorine disinfectant rapidly kills coliform group and leaves chlorine resistant bacteria unaffected [26]. Moreover, Jin et al., (2020) demonstrated that Eterococcus faecalis can survive and highly resistance to chlorine exposure compared to fully sensitive Escherichia coli [10].
The majority of bacterial contamination has been detected in swimming pools Sp1, Sp6, Sp4, and Sp3 with 11 (18.64%), 10 (16.94%), 9 (15.25%), and 8 (13.55%) isolates, respectively. However, the lowest bacterial isolation was found in Sp5, Sp7, Sp8, and Sp2 with 6 (10.16%), 6 (10.16%), 5 (8.50%), and 4 (6.80%) isolates, respectively (Table 1).
Immense range of contamination was found from in the results of bacteriological analysis of public swimming pools water in Sulaymaniyah city. The contamination in water of public swimming pools have been recognized globally such as (17.22%) in indoor pools of Yazd City in Iran [41], (32.9%) indoor swimming pools in Northwestern Greece [42], (68%) swimming pools around Kampala City in Uganda [43], and (18%) of studied pools in Biała Podlaska were above the permissible value [44].
3.4. The impact of water resources and free chlorine on bacterial load in swimming pools:
The rate of water contamination might be resulted from the source of water that are used for the swimming pools including mixed water from well water and water distribution system, mixed water from water tanker supply and water distribution system, well water, water distribution system) with 20 (33.9%), 19 (32.2%), 16 (27.12%), and 4 (6.77%) isolates, respectively (Fig. 1). Moreover, results of bacterial isolates were showed that the number of isolated bacteria directly depend on chlorine concentration. As it is clear maximum number of bacterial isolates 40 (67.80%) have been recorded at lowest free chlorine concentration (≤ 0.2 mg L-1) in swimming pools, followed by free chlorine concentration (0.3–0.5 mg L-1) and (> 0.5 mg L-1) with 16 (27.12%) and 3 (5.08%) isolates, respectively (Fig. 2).
The high rates of water contamination by non-lactose fermenter bacteria might be attributed several factors including hygienic precautions, filtration system, source of water in swimming pools, contamination from bathers, inadequate pool disinfection, periodic pool cleaning, and poor water exchange in swimming pools. This explanation is supported by research from Bello et al., (2012) who found that the presence of large numbers of coliform load in pool water suggests either insufficient chlorination or insufficient protection of the source of untreated water [45].
Additionally, high percentage of contamination of might be resulted from pool filled with water from the well, water tanker, and distribution water system water. The contaminants might be introduced through leakages in pipe, regrowth due to prolong storage of water, contaminated pumps and sanitation systems [52]. Other study also confirmed that distribution system water can easily get contamination by fecal coliforms from the leakage and throughout water supply networks [25]. Moreover, Bartram et al., (2014) described that unprotected springs, surface water, dug wells, tanker trucks are considered not suitable and unimproved water for use [47]. The presence of a high level of organic matter, high level of microbial load, high temperature, and inadequate chlorine concentration might be other potential contributing factors related to pools contamination. It was suggested that organic materials from swimmer’s urine and sweat, temperature, chemical components of water are directly reducing the efficiency and decreases the level of free chlorine in the water [48].
The results of proportional increasing of bacterial contamination in swimming pool with lower chlorine concentration is supported by Czeczelewski (1994) who claimed that the swimmers are under the risk of infectious diseases when free chlorine concentration is insufficient to kill the bacteria in the pool [44]. Moreover, Luo et al., (2018) confirmed that increasing free chlorine concentrations can significantly reduce the frequency of bacterial survival [40].
3.5. Identification of chlorine resistance bacterial isolates:
Results of most probable number (MPN) of all water samples collected during the study were showed negative growth of lactose fermenter bacteria. Therefore, the main objective of this investigation was to isolate and characterize non-lactose fermenter bacteria in MPN negative tests. Results of Vitek-2 compact indicated that all non-lactose fermenter Gram-negative divided on seven Gram-negative bacterial genera including, Enterobacter cloacae ssp. cloacae (11.86%) (7 isolates), E. cloacae ssp. dissolvens (22.03%) (13 isolates), E. cloacae ssp. hormaechei (23.72%) (14 isolates), E. amnigenus (1.7%) (single isolate), Pseudomonas aeruginosa (13.56) (8 isolates), P. fluorescens (1.7%) (single isolate), P. stutzeri (1.7%) (single isolate), Raoultella ornithinolytica (3.38%) (2 isolates), Burkholderia cepacian (6.77%)(4 isolates), B. multivorans (1.7%) (single isolate), Stenotrophomonas maltophilia (5.1%) (3 isolates), Acinnetobacter sp. (1.7%) (single isolate), Acinnetobacter pittii (3.38%) (2 isolates), Atlantibacter hermannii (1.7%) (single isolate) (Fig. 3). Molecular identification by PCR amplification was used for confirmation of identification. The sequence analysis of the amplified 16S rDNA revealed that Enterobacter cloacae ssp. hormaechei (with an accession number OQ421629), E. cloacae ssp. cloacae (accession number OQ401393), E. cloacae ssp. dissolvens (accession number OQ401398, Pseudomonas aeruginosa (accession number OQ401394), P. fluorescens (accession number OQ421630), Stenotrophomonas maltophilia (accession number OQ401399).
Although, chlorine resistant bacteria are a global issue. Therefore, this study provides insight to find the impact of different chlorine concentrations (0, 0.07, 0.15, 0.30, 0.60,.1.25, 2.5, 5 mg L− 1) on growth and biofilm formation of all isolated bacteria. Results of chlorine resistance test (MIC and MBC) showed that all E. cloacae isolates resistance to more than 5 mg L− 1 chlorine, followed by P. aeruginosa, P. stutzeri, Raoultella ornithinolytica, Burkholderia cepacia, Acinetobacter pittii, Atlantibacter hermannii resistance to 5 mg L− 1 chlorine, Enterobacter amnigenus, P. fluorescencea, and Stenotrophomonas maltophilia resistance to 2.5 mg L− 1 chlorine, and Burkholderia multivorans to 1.25 mg L− 1 chlorine.
Results of correlation between different chlorine concentration and growth of isolated bacteria were exhibited linear negative correlation in E. cloacae ssp. cloacae, E. cloacae ssp. dissolvens, E. cloacae ssp. hormaechei, E. amnigenus, P. aeruginosa, P. fluorescence, Raoultella ornithinolytica, Stenotrophomonas maltophilia, Acinetobacter sp., Acinetobacter pittii, Atlantibacter hermannii ranging between (r =-0.74 to -0.82) (Table 3) (Fig. 3) (p < 0.05). However, non-linear weak negative correlation was observed in Pseudomonas stutzeri (r=-0.17), Burkholderia cepacia (r=-0.17), and Burkholderia multivorans (r=-0.54) (Table 3) (Fig. 4) (p > 0.05).
On the other hand, moderate negative linear correlation ranging between (r = -0.72 to r = -0.81) (p < 0.05) (Table 3) was found between different concentrations of chlorine and biofilm formation of E. cloacae ssp. dissolvens, E. cloacae ssp. hormaechei, E. amnigenus, Burkholderia cepacian, Burkholderia multivorans, Stenotrophomonas maltophilia, Acinetobacter sp., Acinetobacter pittii, Atlantibacter pittii. Moreover, weak to moderate negative non-linear correlation between the effect of different chlorine concentrations and growth was determined in each of Enterobacter cloacae ssp. cloacae, Pseudomonas aeruginosa, Pseudomonas fluorescence, Pseudomonas stutzeri, Raoultella ornithinolytica, and Atlantibacter hermannii (r = -0.13 to r = -0.68) (Table 3) (Fig. 4) (p > 0.05).
Most importantly, strong positive linear correlation ranging between (0.90 to 0.98) was recorded between growth and biofilm formation of E. cloacae ssp. cloacae, E. cloacae ssp. dissolvens, E. cloacae ssp. hormaechei, E. amnigenus, Stenotrophomonas maltophilia, Acinetobacter sp., Acinetobacter pittii (Fig. 4) (Table 3) (p < 0.05). However, other isolates P. aeruginosa, P. fluorescence, P. stutzeri, Raoultella ornithinolytica, B. cepacia, B. multivorans, and A. hermannii exhibited weak positive non-linear correlation (Table 3) (Fig. 4). The overall results in quantitative biofilm analysis showed that the degree of biofilm formation was positively correlated with the ability of bacteria to grow at different chlorine concentrations. Moreover, both growth and biofilm formation are negatively correlated with the chlorine concentrations.
Table 3
Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of chlorine. Person correlation between different chlorine concentrations with bacterial growth and biofilm formation of isolated bacteria from all swimming pools. Nutrient broth (NB) mixed with different concentrations of chlorine (0, 0.07, 0.15, 0.30, 0.60,.1.25, 2.5, 5 mg L− 1) in 96 microwell plate was used and incubated at 37°C for 18 hrs. Biofilm formation estimated by the crystal violet staining method. P < 0.05 used as a level of significant.
Bacterial stains
|
MIC & MBC of chlorine
(mg L− 1)
|
Chlorine
vs.
Growth
|
Chlorine
vs.
Biofilm
|
Growth
vs.
Biofilm
|
Enterobacter cloacae ssp. cloacae
|
> 5
|
-0.79*
|
-0.68 ns
|
0.81*
|
Enterobacter cloacae ssp. dissolvens
|
> 5
|
-0.77*
|
-0.79*
|
0.98***
|
Enterobacter cloacae ssp. hormaechei
|
> 5
|
-0.80*
|
-0.80*
|
0.98***
|
Enterobacter amnigenus
|
2.5
|
-0.74*
|
-0.80*
|
0.92***
|
Pseudomonas aeruginosa
|
5
|
-0.75*
|
-0.52 ns
|
0.55ns
|
Pseudomonas fluorescence
|
2.5
|
-0.81*
|
-0.61 ns
|
0.71ns
|
Pseudomonas stutzeri
|
5
|
-0.17 ns
|
-0.16 ns
|
0.26ns
|
Raoultella ornithinolytica
|
5
|
-0.80*
|
-0.63 ns
|
0.61ns
|
Burkholderia cepacia
|
5
|
-0.17 ns
|
-0.72*
|
0.33ns
|
Burkholderia multivorans
|
1.25
|
-0.54 ns
|
-0.75*
|
0.70ns
|
Stenotrophomonas maltophilia
|
2.5
|
-0.77*
|
-0.81*
|
0.94***
|
Acinetobacter sp.
|
5
|
-0.82*
|
-0.80*
|
0.94***
|
Acinetobacter pittii
|
5
|
-0.79*
|
-0.78*
|
0.90***
|
Atlantibacter hermannii
|
5
|
-0.75*
|
-0.13 ns
|
0.09ns
|
Results of current study demonstrated the presence of free chlorine in variable concentrations, which was significantly lower than the permissible level in the whole water of swimming pools. Previous study demonstrated that the application of adequate disinfectants effectively stops the transmission of chlorine-sensitive pathogens [49]. According to WHO (2006), good filtration, and adequate free chlorine level of (1 mg/L) throughout the pool are critically required for public and semi-public swimming pools [38]. Moreover, to control bacterial survival in the pools Yahya et al.(1990), confirmed that chlorine disinfectant needs to be supplied in a high quantities and through all the times [51].
Some of Gram-negative bacteria isolated in this study were belong to non-pathogenic, opportunistic, and common pathogen to human. The high degree of contamination in swimming pool water posed a possible hazard to man [54]. In most countries, chlorination is used as the main water treatment method in swimming pools[10]. However, the risk of chlorine and antibiotic resistance bacteria cannot be completely controlled [52]. According to the previous study conducted in Sulaymaniyah city, microbiological content of water samples did not reveal the existence of fecal coliform bacteria. However, non-lactose fermenter chlorine resistant Enterobacter cloacae was found to be considerably determined[20].
Obtained results of this study showed that Enterobacter cloacae 57.5% (34/59) and Pseudomonas aeruginosa 13.56% (8/59) are most commonly isolated bacteria from swimming pools (Table 1). The absence of international bacterial indicators of water quality such as total and fecal coliform E. coli might be due to the efficiency of chlorination to eliminate chlorine sensitive fecal coliform and survival of chlorine resistant Enterobacter spp. and Citrobacter freundii [26, 53]. It has been documented that chlorine disinfectant failed to inactivate chlorine resistance E. cloacae[20], Pseudomonas sp. [54], Acinetobacter [55] in water. Several studies have reported the ability microorganisms to survive in chlorinated water [20, 58, 59, 60]. The prevalence of Enterobacter cloacae in chlorinated swimming water is consistent with the previous study by Najmuldeen(2021), who isolated (56%) chlorine resistant E. cloacae from chlorinated water storage tanks [20]. Other study also isolated P. aeruginosa from swimming pool water that causes, wound infection, respiratory diseases, otitis media in ears swimmers [58, 59].
The usage of irregular quantities of chlorine-containing compounds in swimming pools may be directly related to the emergence of bacterial isolates with hyper chlorine resistance in Sulaymaniyah public pools. Environmental isolates that have previously been exposed to chlorine may become resistant to disinfectants [15]. Furthermore, additional research showed that the excessive and frequent usage of sodium hypochlorite has led to the emergence of chlorine-resistant microorganisms (NaOCL) [16, 60].
Additionally, the survival of waterborne opportunistic pathogens in treated water with disinfectants linked to several mechanisms including modification in cell surface, attachment to surfaces or particles, exopolysaccharide barrier, biofilm formation, efflux pump activity, and spore formation[1, 53, 61, 62], potential reduction in cell permeability [15], a number of genes that regulate oxidative stress, DNA repair, pore protein regulation, and cell wall repair have a protective role [52].
3.6. Bacterial Antibiotic Resistant test
The most significant factor impacting the level of antibiotic resistance in treated water samples was revealed to be residual chlorine. Therefore, all isolated bacteria in this study were subjected to antibiotic sensitivity test for 15 different antibiotics using disc diffusion method recommended by CLSI-guidelines (CLSI-2013).
The obtained results demonstrated that relatively all bacterial isolates were resistant to cefotaxime β-lactam antibiotics of third generation cephalosporins, amikacin, aminoglycoside antibiotic, similarly resistant to other classes of antibiotics including Nitrofurantoin, Erythromycin. Likewise, with the exception to few isolates of Enterobacter cloacae, resistant to additional antibiotics with variable mode of actions such as minocycline second generation of tetracycline, chloramphenicol, nalidixic acid was recorded (Fig. 5). However, levofloxacin, tobramycin, and ciprofloxacin were exhibited the most effective antibiotics against all isolated bacteria except, few strains of P. aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia (Fig. 5). Finally, a variable pattern of resistance and sensitivity were noticed to imipenem, meropenem, ceftazidime, ceftriaxone, gentamicin antibiotics (Fig. 5).
Antibiogram analysis indicated that isolated bacteria belong to Burkholderia cepacia, P. aeruginosa, Stenotrophomonas maltophilia, E. cloacae spp. cloacae, E. cloacae spp. dissolvens, and E. cloacae spp. hormeichae are resistant to most of applied antibiotics. However, the most sensitive bacterial isolates to more than four antibiotics classes are belonging to E. amnigenus, P. fluorescens, P. stutzeri, Raoultella ornithinolytica, Acinetobacter sp. Acinetobacter pittii, and Atlantibacter hermannii, Burkholderia multivorans.
There is yet no conclusive physiological link between the mode of antibiotic action and resistance to chlorine disinfectants. Recent study suggested that aquatic environment could serve as an important reservoir of antibiotic resistant genes (ARGs) that horizontally transmitted to other closely related bacterial species [64]. It has been hypothesized that chlorination encourages the transformation of plasmids among bacteria in their natural environment, which promotes the development of antibiotic resistance strains [31, 65]. This concept has been proven by Jin et al,(2020), who stated that chlorination process enhances natural transformation of plasmid, which led to the exchange of ARGs between bacterial genera and emergence of new antibiotic resistant bacteria (ARB) [10].
In agreement with this finding, other studies have verified that E. cloacae susceptible to levofloxacin, ciprofloxacin, and resistant to nitrofurantoin and cefotaxime [66, 67]. Previous study documented that E. cloacae possesses high capacity to acquire genes encoding resistance to numerous classes of antibiotics, including β-lactamase inhibitor, aminoglycoside, tetracycline, and carbapenem [66, 68]. Similarly, 96% of isolated P. aeruginosa from swimming pools have shown multidrug resistant [71]. B. cepacia complex strains can survive for long durations in water, disinfectant and exhibited resistant to a wide range of antimicrobial drugs, including polymyxin, aminoglycosides, carboxypenicillins, first and second generation cephalosporins [70, 71]. Additionally, Govender et al. (2020), stated that presence of multidrug resistant Acinetobacter spp. and Stenotrophomonas maltophilia in water increases the probabilities of community acquired infections [72]. Maintaining safe pool water quality is crucial to prevent bathers' health problems. Regular water quality checks are necessary to avoid these undesirable effects related to contamination of swimming pools. The findings of this study supported the existence of waterborne pathogens that may cause a risk for various bacterial illnesses.