By Nohelia Castro-del Campo, Ph.D. and Cristóbal Chaidez-Quiroz, Ph.D.

Consumption of bottled, non-carbonated water in Mexico has greatly increased over the past decade, due to its convenience and perceived relative safety. This increase has been attributed to consumers’ escalating concern over elevated tap water pollution, and objections to offensive tastes or odors, chlorine or other additives from municipal water supplies (Warburton and Dodds, 1992). Along with the significant increase in bottled water consumption, there is growing concern over the microbiological quality of such products.30

Drinking water stations (DWS) were introduced into the Mexican market as another consumer option to bottled drinking water. DWS are located at convenient locations, near highly populated urban and rural areas where the water bottle, provided by the consumer, is only washed with soap and rinsed with tap water before being filled. Consumers bring their own 20-liter (5.28-gallon) containers and water is dispensed by a dispatcher. The question arises as to whether or not the water is hygienically produced and dispensed, especially where the lack of training in hygienic practices is considered.

In Mexico, water from both bottling companies and water stations must meet the Mexican federal quality standard required for drinking water (NOM-041-SSA1-1993). Drinking water is any potable water that is further treated (generally by carbon filtration, reverse osmosis and/or ultraviolet light) prior to bottling and sealed in food-grade bottles. Bacteriological quality of bottled water is based on the presence/absence of indicator bacteria for fecal contamination (Escherichia coli), surface water contaminants (total coliforms) and opportunistic bacterial pathogens such as Pseudomonas aeruginosa.20 Indigenous bacteria remain at low numbers while the water is in its natural environment but a rapid growth develops after water is bottled. The reason for this altered-growth behavior is not clear, but it may be due to oxygenation of water during the bottling process, the increased surface area provided by the bottle and trace amounts of nutrients arising from the bottle.32 Even more, the bottled water is chlorine free, which provides the opportunity for bacteria (if present) to grow.

Study goals
Mexican and international regulations suggest that total coliforms and Pseudomonas aeruginosa might be absent and HPC bacterial counts should not exceed 500 colony-forming units (CFU) mL-1, primarily because of the interference of coliform detection (NOM-041-SSA1-1993).31,37 The purpose of the study was to evaluate the bacteriological quality of bottled water in Culiacán, Mexico: to compare the quality of bottled water sold in local supermarkets with that of bottled water delivered directly to households, and to evaluate the importance of handling factors, such as cleanliness and storage time.

Materials and methods
Sampling. For the purpose of this study, drinking water fell into two categories: (a) 2-liter (0.53-gallon) bottled water (2-LBW) sold in local supermarkets (national and international brands) and (b) water from local water stations dispensed in 20-liter (5.28-gallon) bottles (20-LBW). Nine different brands were analyzed corresponding to category (a); while for category (b), seven brands were 20-liter, machine-filled bottled water (MFBW) and four brands were 20-liter, hand-filled bottled water (HFBW). Samples of MFBW were obtained from bottles in household settings with previous consent of the homeowners, while HFBW samples were obtained directly from the drinking water filling station. During a six-week period, a total of 120 water samples were collected for conducting the experiment. Water samples were placed in sterile 1-liter (0.26-gallon) plastic bottles containing 2 mL (0.068 fluid ounces) of sterile 10-percent sodium thiosulphate to neutralize any residual chlorine. Samples were transported refrigerated to the Environmental and Food Microbiology Laboratory of Centro de Investigacion en Alimentacion y Desarrollo-CIAD Research Center for Food and Development Culiacán Station for processing within one hour after collection.

Microbial detection. P. aeruginosa and total coliforms in water samples were enumerated (counted) using Pseudomonas agar and m-Endo, respectively. One 100-mL sample was tested for total coliform bacteria, one 100-mL sample for P. aeruginosa, and one 1-mL sample for HPC bacteria. P. aeruginosa and total coliform samples were incubated at 37°C (98.6 °F) for 24 hours. HPC bacteria were enumerated and incubated at 37°C for 24 hours. Presumptive identification of P. aeruginosa was based on the production of green pigmentation (the Oxidase Test).

Biochemical identification. Total coliforms isolated were confirmed using the API20E system (Biomerieux Vitek, Hazelwood, MO), a system which can biochemically identify the type of organism. Two selected colonies from each week were subjected to biochemical identification.

Statistical analysis. The comparison of the two different drinking water sources was made based on the Chi Square statistical analysis.21

Results and discussion
From a total of 120 bottled water samples analyzed, 65 (54 percent) were 20-LBW and 55 (46 percent) were 2-LBW. The number of samples in which the studied bacteria were detected is shown in Tables 1 through 4. The bottled water tested in the study was processed with UV, carbon filters and RO, and the source water came from an approved local water supply system. The health authorities of Mexico coincided with the US EPA requirements, which suggests that HPC bacteria should not exceed 500 CFU/mL in drinking water due to several inconveniences such as interference with the detection of coliform indicators or pathogenic bacteria.4

Total coliforms. Total coliforms were detected in a range from 0.75 CFU/100 mL to 2.3 x 104 CFU/100 mL in hand-filled containers (20L-HFBW), while from 2.10 CFU/100 mL to 508 CFU/100 mL for machine-filled containers (20L-MFBW), and finally from 0.11 CFU/100 mL to 0.33 CFU/100 mL for local supermarket containers (2-LBW) (see Table 1). Statistical significances (P < 0.05) between 20-LBW and 2-LBW were detected. Non-compliance due to the presence of total coliforms was higher than reports elsewhere32, 2; however, the 20-LBW analyzed in the current study were delivered to households by trucks with no refrigeration, which may have had an effect on the prevalence of total coliforms, P. aeruginosa and HPC bacteria. It has been previously reported that five-gallon bottled water analyzed exceeded the limits established by Mexican standards.25 The absence of verified E. coli in the 120 samples tested supports earlier indications that this bacterial pathogen is rare in bottled water15; however, other genus of the coliform group was frequently identified.

Pseudomonas aeruginosa. The average concentration and maximum values for P. aeruginosa is shown in Table 2. In general, P. aeruginosa was detected in 23 of the 120 samples (19.16 percent) collected during the six-week study. Individually, 2-LBW represented 11.11 percent (6/54), HFBW 25 percent (6/24) and MFBW 26.19 percent (11/42). Bacterial concentrations ranged from 0 CFU/100 mL to 5.6 x 105 CFU/100 mL in HFBW, while for MFBW, the counts ranged from 0 CFU/100 mL to 2.1 x 105 CFU/100 mL. Finally, the minimum concentration detected for 2-LBW was 0 CFU/100 mL with a maximum of 46 CFU/100 mL. These results show high concentrations of this opportunistic pathogen. Organizations such as the World Health Organization established that water must be free of Pseudomonas aeruginosa because of the risk it could present to the most susceptible stratums of the population (children and immunocompromised people). Correlation and regression analyses were performed on bacteriological data; however, no significant correlation was found (data not shown).

The International Bottled Water Association states that bottled water must be free of coliforms (0/100 mL) and P. aeruginosa (0/100 mL).17 In the current study, P. aeruginosa was present in 19.16 percent and total coliforms in 15.83 percent of the total samples. Opportunistic bacterial pathogens were detected in the bottled waters (see Table 2). The results are similar to those reported by other authors.8, 22, 9, 13, 30, 16, 18, 6 Warburton et al. (1994) mentioned that this organism is able to survive and proliferate to levels of 1,000 CFU/mL in bottled water stored at room temperature. Bacterial contamination can be attributed to several factors, including contamination during bottling and storage.11 Prolonged storage at room and refrigeration temperatures allows multiplication of bacteria to numbers >1 x 105. Several authors determined that the risk of colonization (not necessarily illness) for ingestion of P. aeruginosa for a daily exposure of 38,000 organisms in two liters of water was 1.4 x 10-6 in healthy individuals.3, 12, 33, 26 The highest concentration of P. aeruginosa observed in this study was in 20-LBW with an average of 1356 CFU/100 mL. Thus, the probability of colonization of the gut by a one-time exposure to this organism from the water samples analyzed in this study is still low. The role of multiple exposures and the ability of these organisms to colonize the gut are currently unknown but could lead to a greater probability of the gut being colonized. It should also be kept in mind that the models used by Rusin et al. (1997a) predict colonization only and not disease. It is unclear whether colonization of the intestinal tract is a prerequisite for illness by these organisms. Based on a daily ingestion of two liters of bottled water, P. aeruginosa had a probability of less than 10-6 of colonizing the gut. If an average person in Mexico consumes a mean of 1.15 L per day as bottled water, then 2.45 x 1010 HPC would be consumed per week. On a weekly basis, the average consumer in Mexico (if one consumes bottled water) ingests less than five percent of his total bacterial intake from bottled water.

Heterotrophic plate count. The average count for heterotrophic bacteria was 1,001-10,000 CFU/mL in the three water categories. Concentrations fluctuated around four-log10 but a slight difference (not statistically significant) was observed among water categories, with MFBW (5.4 x 104 CFU/mL) having higher contamination, followed by HFBW (5.2 x 104 CFU/mL) and finally 2-LBW (2.8 x 104 CFU/mL) (see Table 3). The greatest concentration of HPC bacteria was found in 20-LBW and the lowest in 2-LBW (see Table 1). The increase in HPC bacteria in 20-LBW is probably due to the study area’s tropical climate, which favors rapid bacterial growth caused by handling of bottles (which are constantly exposed to irradiation and increasing water temperature) before being delivered, and the lack of bottle disinfection practices, especially hand-filled, which are only washed with soap and rinsed with tap water before being filled. Culiacán is characterized by ambient temperatures frequently above 35°C (89.6°F). Also, the period of time from bottling to delivery could take all day in mostly non-refrigerated trucks. Additionally, the filling process in HFBW stations was deficient, since no disinfection was applied to the 20-L bottle before filling.

Both Mexican and international regulations have suggested that HPC bacteria counts in drinking water should not exceed 500 CFU/mL, primarily because of interference with coliform detection (NOM-041-SSA-1993).31, 37 In this study, drinking water often failed to meet such a recommendation. HPC bacteria were often present in numbers that ranged from 1 x 103 to 1 x 107 CFU/mL. It has been found in previous studies that the bacterial population of bottled water increases after bottling, reaching a peak between the first and second week13 and remains reasonably constant for at least six months. In the current study, a large variation was found in the colony counts at different sell-by dates, both within and between brands, resulting in generalizations about shelf life and the difficulty in maintaining a constant microbiological quality. In the present study, the HPC bacteria counts were higher in 20-LBW than 2-LBW.

Caution is needed when interpreting the public health significance of HPC bacteria in drinking water. Although it has been suggested that these bacteria may be potentially pathogenic to vulnerable individuals, the potential for adverse health effects appears to be low.26 The occurrence of HPC bacteria in bottled water is a major factor in preventing the growth of waterborne bacteria, such as Salmonella. Thus, sterile water is not necessarily desired.5 Gerba et al. (2002) stated that HPC intake from water sources remains low when compared with that of food intake. Although the species of bacteria found in water reflects the same as found in food items, the exposure via foods and other environmental sources is far more significant than bottled and other drinking water sources.

Heterotrophic-plate-count bacteria by themselves do not present a risk to consumers. High numbers in a distribution system may represent a contamination or another water quality problem; however, this cannot be quantitatively related to an illness risk.27 The occurrence of specific bacteria within the HPC population, however, could present a potential risk if the numbers are great enough. Thus, HPC bacteria in bottled water do not represent a significant source of HPC bacteria in the average diet of consumers in Mexico.

Biochemical identification. Identification of isolated coliforms is shown in Table 4. This identification showed the constant presence of the genus Klebsiella. Flavobacterium, Moraxella and Enterobacter were also detected. Identification of the strains isolated from HFBW and MFBW showed higher genus variability than those isolated from 2-LBW. Moraxella and Flavobacterium were the only genus identified in 2-LBW; Klebsiella pneumoniae pneumoniae, Klebsiella oxytoca and Acinetobacter baumannii calcoaceticus were only isolated from MFBW.

The microbiological quality of bottled drinking water in Mexico was found to be acceptable. The contamination with pathogenic and opportunistic microorganisms seemed to occur within household settings, even though HPC bacteria were present in bottled water that did not represent a significant source of HPC bacteria in the average diet of consumers in Mexico. Purification processes need to be reformed in order to eliminate the presence of opportunistic pathogens in drinking water in Mexico.

This study was supported by the Consejo Nacional de Ciencia y Tecnología, México, Project #35558-B. The authors thank MS José Gabriel Cazarez Diarte for his valuable technical support.


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About the authors
Cristobal Chaidez-Quiroz, Ph.D. is a scientist and Regional Director of the Research Center for Food and Development (Centro de Investigacion en Alimentacion y Desarrollo-CIAD) in Culiacan station. He also teaches graduate and undergraduate courses at CIAD and the Autonomous University of Sinaloa (AUS), respectively. Chaidez-Quiroz earned a Bachelor’s Degree from the School of Biology, Chemistry and Pharmaceutical Sciences (AUS), with a major degree in biology and minor in chemistry. He earned his Doctorate at the University of Arizona, under the direction of Dr. Charles P. Gerba, and can be reached via email at [email protected].

Nohelia Castro-del Campo, Ph.D. is a scientist and Professor of the Research Center for Food and Development (Centro de Investigacion en Alimentacion y Desarrollo-CIAD) in Culiacan station. She earned her Doctorate Degree from the University of Arizona, also under the direction of Dr. Charles P. Gerba. She is member of the National Scientists System of Mexico and can be reached via email at [email protected].


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