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Potential Salmonella transmission from ornamental fountains. (Features).


At different levels of frequency and severity, both developed and underdeveloped countries are still affected by outbreaks and individual cases of waterborne diseases. In the United States between 1986 and 1988, 50 outbreaks and 25,846 recorded cases were associated with ingestion of water contaminated with protozoa, bacteria, viruses or chemical substances, or of unknown etiology (Levine, Stephenson, & Graun, 1991). Important improvements in water system sources, treatment, storage, operation and maintenance of distribution networks, and overall management have helped reduce these numbers to only 17 outbreaks and 2,038 cases during 1997-1998 (Communicable Disease Centers, 2000).

Water shortage or unavailability is an implicit risk factor in public health. Improvements in water supply alone may lead to a significant reduction in diarrheal morbidity A water supply soon reduces the prevalence of Shigella carriers in a community, even if the water does not receive any type of treatment (Hollister, Beck, Gittelsohm, & Hemphill, 1955). On the other hand, control of typhoid fever or cholera transmitted by water depends primarily on improving the microbial quality of the water.

Certain human activities may be contributing factors in the transmission of waterborne diseases. Some communities use raw sewage to irrigate crops, whereas in other cases, ignorance, tradition, or irresponsibility may in several ways lead to use of poor-quality water that contaminates food. For instance, street vendors sometimes spray sliced fruits with the same water they have used to rinse their hands and the external parts of the fruits before peeling. The source of that water may be, when the opportunity exists, any recreational fountain near the vendor. These fountains may be located in city squares, parks, and other places to which animals such as dogs and pigeons have direct access. Other types of recreational waters exposed to animals have been reported as a source of infection in cases of dermatitis, gastroenteritis, meningitis, Pontiac fever, and leptospirosis (Levine et al., 1991).

Although the oral route of infection is the most common, the respiratory route may also be involved (Newman & Foran, 1992). While one can prevent health risks like those mentioned above by avoiding direct contact with contaminated water and by not consuming the foods involved, there is another exposure risk for people who stay or walk around the fountains. When the fountain water is sprayed into the atmosphere, particularly under windy conditions, inhalation of droplets may lead to infections caused by pathogenic microorganisms contained in the water.

In this study, the authors measured the presence of Escherichia coli and Salmonella in water samples obtained from ornamental fountains located in Guadalajara City.


From five ornamental fountains located in Guadalajara city squares, 145 water samples were obtained over two periods (six and 10 months). In general, the water appeared clean and clear. Samples were collected with a Moore swab (Moore, Sagik, & Sorber, 1979) made by folding cotton gauze to form a pad of 20 centimeters (cm) by 6 cm. The swabs were tied with twine, wrapped in aluminum foil and autoclaved. For sampling of water, a swab was suspended in the fountain, tied between a brick and a floating piece of previously sterilized wood, at approximately 20 cm from both the bottom and the surface of the basin. The swabs were left in place for 24 hours as the water was propelled by the fountain's pumping system.

Microbiological Analysis

After the 24-hour sampling time, the swabs were removed from the water, drained, and separated from the attached floating parts. Each was placed in a sterile plastic bag and transported to the laboratory; analysis was begun within two hours. During the first sampling period, 45 swabs were collected and tested only for Salmonella. Tetrathionate brilliant green broth (TT, 100 mL) was added to the bag containing the swab, which was incubated at 35[degrees]C for 24 hours.

During the second sampling period, 100 samples were collected from the same fountains sampled in the first period, but each sample consisted of two swabs tied together and held submerged in the water as described above. A sample of water was also collected on each occasion in a Pyrex glass bottle containing sodium thiosulfate as a chlorine neutralizer. This water was used to quantify coliform bacteria and E. coli. One drained swab was transferred to 100 mL of TT broth and the other to 100 mL of selenite cystine broth. Enrichment media were incubated at 43[degrees]C for 24 hours. To isolate and identify Salmonella, each broth was streaked on bismuth sulfite agar, Salmonella-Shigella (55) agar, xylose-lysine-deoxycholate (XLD) agar, and brilliant green sulfadiazine agar, followed by screening on triple-sugar iron agar (TSI) and lysine iron agar (LIA) and identification of serovars with somatic and flagellar antisera. In the first study, only one isolate was serotyped per positive sample; in the second study, 1 6 to 22 colonies (when available) were tested. The coliform count was conducted by the most-probable-number (MPN) method in lactose broth by inoculation with 10 mL of water (in double-strength broth), and 1, 0.1, and 0.01 mL of water (in single-strength broth). Tubes showing gas production were confirmed in brilliant green lactose broth (BGLB). For E. coli identification, positive tubes of BGLB were streaked onto eosin methylene blue (EMB) agar, two to three colonies per sample were tested for indol production in tryptose broth and for lactose fermentation in lactose broth (both after incubation at 44[+ or -] 0.2[degrees]C) (Mossel & Moreno Garcia, 1982).

All media used were from BIOXON/Becton Dickinson (Mexico), and antisera were kindly provided by the National Institute of Diagnosis and Epidemiological Reference in Mexico.

Results and Discussion

Salmonella is an intestinal bacterium excreted in feces by a diverse range of animals. The ubiquity of Salmonella spp. in the natural environment favors water contamination. The principal theme of this study was demonstration that ornamental fountains, which attract people, have the potential to generate Salmonella-containing aerosols. The study further suggests that, since aerosol-transmitted infections with this pathogen have been demonstrated in various animals, such transmission might also affect humans. Salmonella enterica was recovered from nine (20 percent) of the samples during the first period and nine (9 percent) during the second period. Although municipal water in Guadalajara city is regularly chlorinated, water recirculating in fountains is not. Since the detection technique for Salmonella in the second period included two enrichment broths, elevated incubation temperature, and a larger sample size, the diminished isolation percentage must have resulted from other, external factors. No changes in the operation of the fountains during the study could be found to explain this difference. In any case, the presence of this pathogen in the ornamental water sources is of concern.

Incubation of tetrathionate brilliant green broth at selective temperatures (41 to 43[degrees]C), rather than permissive temperatures (35 to 37[degrees]C) markedly increases the sensitivity of the method by facilitating isolation of Salmonella on plating media. The elevated temperature enhances the selectivity of the medium against competing flora. Selenite cystine is generally incubated at 35 to 37[degrees]C because selenite-based media can be inhibitory to Salmonella at higher temperatures. A minimum of two enrichment media and two incubation temperatures always should be used to optimize the detection of contaminated samples.

The S. enterica serovars isolated from the fountains are listed in Table 1. Twelve (25 percent) of the positive Salmonella samples contained more than one serovar. The numbers of different serovars isolated from Salmonella-positive samples were as follows: two (in two samples), three (in one sample), four (in one sample), and five (in two samples). S. Typhimurium was identified in six (33 percent) of the samples that were positive for Salmonella. This serovar has been traditionally demonstrated to be from human sources in Mexico, although recent trends indicate that S. Enteritidis also is increasingly being isolated from human cases (Gutierrez-Coggo, Gonzalez-Bonilla, Giono-Cerezo, & Beltran, 1994). Other serovars recovered in this study, such as S. Agona, are also present in human and food sources. Interestingly, S. Derby, which is the serovar most commonly isolated from foods in Mexico and is also an important serovar among human infections, was absent in this study. This result would indicate differences between the sources of Salmonella contamination in ornamental-fountain water and the common sources of Salmonella in foods and drinking water. In addition, the survival times of different serovars may determine whether they can be recovered from water. S. Typhimurium has been reported to survive for 120 days in tap water, while S. Dublin survived for 87 days (U.S. Department of Agriculture & Food and Drug Administration, 1969).

People, dogs, and birds, especially pigeons, were seen to have direct access to the fountains. This human and animal contact may be a route by which pathogenic organisms reach the water and, at the same time, a source of Salmonella infections if the water is ingested or touched. In addition, contaminated fountain water may increase the risk of airborne transmission of pathogens.

Although Salmonella was present in only a small number of Moore swabs during the second sampling period, coliform organisms were detected in 75 (75 percent) of the water samples, at concentrations ranging from 1 to >10 colony-forming units per milliliter (CFU/mL) (Table 2). Several authors question the accuracy of using coliform organisms to assess the safety of water or foods, arguing that their presence does not necessarily indicate any antecedent of fecal contamination. To determine whether coliform counts correlated with fecal contamination, tests for E. coli also were carried out in the water samples. E. coli is currently considered to be the best indicator of fecal contamination in foods and water (Mossel, Corry, Struijk, & Baird, 1995). The data given in Table 2 indicate that E. coli was absent in as much as 44 percent of the samples that had tested positive for coliforms. On the other hand, E. coli was isolated from seven (28 percent) of the samples that were negative for coliforms. Statistical analy sis (Chi-square test) showed that there is a correlation between the presence of coliforms and E. coli in the 100 samples analyzed from water fountains (p < .05). The presence of animals in the area and above the fountains that were sampled is consistent with the finding of F. coli and coliform bacteria in the water. Thus, fecal contamination could be considered an important factor in the sanitary quality of the ornamental fountains studied for this research.

The water in these fountains is projected several meters high, and large aerosols may be formed in windy weather. Although this is a beautiful spectacle, the aerosol may be a source of bacterial pathogens or parasite cysts. If solid matter is suspended in the water, an aerosol of droplet nuclei will persist after evaporation and may continue to be infective (Darlowe & Bale, 1959). Moreover, exposure to water in a Minnesota water sprinkler fountain was associated with an outbreak of cryptosporidiosis (Centers for Disease Control and Prevention [CDC], 1998). Eleven children out of 120 visitors met the case definition. The implicated fountain comprised 14 nozzles that spurted jets of water vertically approximately 1 to 6 feet (30 to 180 cm). In the description of this outbreak, water inhalation was not considered as a possible source of the parasite. Airborne salmonellosis and other enteric diseases have, however, been reported. Netter (1950) described an outbreak of salmonellosis in a pediatric hospital, where the serovars involved, S. Oranienburg and S. Choleraesuis, were first isolated from the upper-respiratory tracts of two infants, who later developed gastrointestinal symptoms. These serovars were isolated from other cases in the same area of the hospital, which suggested that the first case was the source of transmission to the other cases through airborne cross-infection. Parasitic diseases such as cryptosporidiosis have been reported to develop after inhalation of oocysts (CDC). Isolation of intestinal pathogens such as Salmonella and E. coli from the upper-respiratory tract has been reported (Darlowe, Bale, & Carter, 1961), Infective hazards by aerosols have been documented in toilets that are flushed without cover (Darlowe & Bale, 1959; Gerba, Wallis, & Melnick, 1975). Other situations where contaminated water can be a vehicle of biological hazards to humans include the use of contaminated water to prepare foods or to wash fruits and vegetables that will be consumed raw, and swimming in contaminated lake s, ponds, bathing beaches, or swimming pools (Van Orden, 1990; Homes, Kinde, Pearson, & Hennes, 1989).

The potential role of airborne transmission of Salmonella is not clearly defined. Some studies have, however, demonstrated that aerosols of at least some Salmonella serovars can infect animals such as calves and mice (Wathes, Zaidan, Pearson, Hinton, & Todd, 1988), chicks (Gast, Mitchell, & Holt, 1998) and laying hens (Baskerville et al., 1992). Doses as low as 150 CFU can be enough to cause infection in mice, as can doses of approximately [10.sup.4] CFU in calves (Waffles et al.). In humans, detailed investigations have shown that the ingestion of just a few Salmonella cells can be infectious. Recent evidence suggests that one to 10 cells can constitute a human infectious dose (D'Aoust, 1994). Since mucus from the respiratory tract is swallowed to a significant extent, it is possible that, especially for immuno-compromised people, inhalation of contaminated aerosols could be a real hazard.


The results of this study suggest that inhalation of mists formed by contaminated water from ornamental water sources, may promote the spread of pathogenic organisms in the population. The access of animals to water fountains, as well as the observed defecation of birds in the water, can be an important source of Salmonella and other pathogenic organisms. Human pathogens have been isolated from bird droppings (Davenport, 1990). Water has been considered a possible reservoir and vehicle of human infections for Plesiomonas shigelloides (Abbey, Emerinwe, Phill, & Amadi, 1993) and is the most common route of infection of Cryptosporidium and Giardia (Smith, 1993). Risk of infections among people who are passively and involuntarily exposed to contaminated aerosols generated by ornamental fountains can be decreased by a strict regimen of disinfection, and by better education about the health risks of having contact with such aerosols.

Coliform Bacteria, Escherichia coli, and Salmonella Serovars in Water
Samples Obtained from Ornamental Fountains

Sample Coliforms (a) E. coli (a) Salmonella enterica Serovars

 1 364 364 S. Agona
 2 0.28 0.28 S. Agona
 3 8.58 1.8 S. Agona
 4 ND (b) ND S. Agona
 5 ND ND S. Anatum
 6 ND ND S. Give
 7 ND ND S. Heidelberg
 8 364 364 S. Ohio
 9 ND ND S. Typhimurium
 10 ND ND S. Typhimurium
 11 ND ND S. Typhimurium
 12 ND ND S. Typhimurium
 13 ND ND S. lyphimurium
 14 8.58 0.28 S. Agona, S. Drypool
 15 1.8 1.8 S. Agona, S. Cerro, S. Enteritidis
 16 8.58 8.58 S. Agona, S. Cerro,
 S. Enteritidis, S. Typhimurium
 17 364 364 S. Aequatoria, S. Agona, S. Cerro,
 Duesseldorf, S. Enteritidis
 18 8.58 364 S. Agona, S. Cerro, S. Duesseldorf,
 S. Enteritidis, S. Worthington

(a)MPN/mL. (b)ND = not done.

Association Between Coliform Recovery and the Presence of Escherichia
coli in Water Samples Collected from Ornamental Fountains (a)

Coliforms Presence of E. coli
 Positive (b) Negative Total

Positive (c) 42 (56%) 33 (44%) 75 (100%)
Negative 7 (28%) 18 (72%) 25
Total 49 51 100

(a)The association between occurrence of coliforms and occurrence of E.
coli is significant (Chi-square test: p < .05).

(b)[greater than or equal to]1 CFU/mL.

(c)[greater than or equal to]0.01 MPN/mL.


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Corresponding Author: Eduardo Fernandez Escartin, Departamento de Investigacion y Posgrado en Alimentos, Facultad de Quimica, Universided Autonoma de Queretaro, Centro Universitario, Cerro de las Campanas, Mexico 76010. E-mail: <>.
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Author:Cliver, Dean O.
Publication:Journal of Environmental Health
Date:Nov 1, 2002
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