Heterotrophic Bacteria in Bottled Water

Heterotrophic Bacteria in Bottled Water

Heterotrophic Bacteria in Bottled Water EN Kokkinakis, Technological Education Institute (T.E.I.) of Crete, Ierapetra, Crete, Greece & 2011 Elsevier B...

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Heterotrophic Bacteria in Bottled Water EN Kokkinakis, Technological Education Institute (T.E.I.) of Crete, Ierapetra, Crete, Greece & 2011 Elsevier B.V. All rights reserved.


Code of Federal Regulations European Community Environmental Protection Agency Food and Drug Administration good manufacturing practice Hazard Analysis and Critical Control Point heterotropic plate count plate count agar polyethylene terephtalate polyvinylchloride

Introduction Bottled water consumption has increased steadily throughout the past decade in both the United States and Europe. It is the second largest commercial beverage category in the United States, with 29 gallons per person, only exceeded by carbonated soft drinks. Nevertheless, US bottled water consumption holds the ninth position worldwide, with United Arab Emirates the first, with 68 gallons per capita annual consumption. Bottled water as an end product rarely contains pathogens unless contamination occurs during bottling, but it may contain heterotropic plate count (HPC) bacteria, which can proliferate during bottling and storage at ambient temperatures. HPC bacteria are those that require organic carbon, rather than carbon dioxide as a carbon source, and all human pathogen bacteria are heterotrophic. European Community (EC) Directive of 1980 suggested that HPC counts in bottled water should be less than 100 colony-forming units (cfu) ml 1 (for HPC at 22 1C) and less than 20 cfu ml 1 (for HPC at 37 1C), whereas the US Environmental Protection Agency (EPA) suggested for HPC bacteria the upper level of 500 cfu ml 1, primarily because of the interference of coliform detection. Bottled water consumed by high-risk categories or shared among individuals can introduce a certain degree of risk due to the presence of opportunistic pathogens that are members of the HPC bacteria group and may possess antibiotic resistance. Nevertheless, the use of good manufacturing practices (GMP) and the correct implementation of Hazard Analysis and Critical Control Point (HACCP) programs in bottling plants can eliminate microbial survival, producing bottled water of good microbial quality.


Bottled Water Definitions and Regulations Bottled water in the United States is regulated by the US Food and Drug Administration (FDA), whereas the tap water is regulated by the EPA. FDA regulates bottled water as a food and has established specific regulations in Title 21 of the Code of Federal Regulations (21 CFR), including standard of identity regulations (21 CFR & 165.110 [a]) that define different types of bottled water and standard of quality regulations (21 CFR & 165.110 [b]) that define levels for contaminants (physical, chemical, microbial, and radiological). FDA has also established GMP regulations, to be followed by bottling companies (21 CFR part 129), labeling regulations (21 CFR part 101), and GMP regulations (21 CFR part 110) for foods in general, that also apply to bottled water. FDA has defined various types of bottled water, such as artesian water (water from a well that taps a confined aquifer in which the water levels stand at some height above the top of the aquifer), mineral water (water with no more than 250 ppm of total dissolved solids that originates from an underground water source (which is geologically and physically protected). Minerals and trace elements should exist at constant levels while addition is not allowed.), purified water (water produced by distillation, deionization, reverse osmosis, or other processes that meet the definition of purified water in the US Pharmacopeia (USP), 23rd revision January 1995), sparkling bottled water (water that after treatment and possible replacement of carbon dioxide contains the same amount of carbon dioxide that it had at emergence from the source), and spring water (water derived from an underground formation by natural flow to the surface of the earth. Spring water may be collected at the spring or through a bore hole tapping the underground formation feeding the spring.). In Europe, three major categories of bottled water can be identified: natural mineral water (underground still or aerated water responding to strict criteria, such as protection against pollution hazards, with a constant level of minerals and trace elements and without any form of treatment or addition of flavors or additives), spring water (underground water protected against pollution hazards, without constant mineral composition and without any form of treatment), and purified water (underground or surface water that has been treated to be suitable for human consumption, the only difference from tap water being the price and packaging in bottles). Directive

Heterotrophic Bacteria in Bottled Water

98/83/EC, in Europe, provides all necessary regulations (physical, chemical, microbiological, and radiological) for water intended for human consumption, including the bottled water. Additionally, the Directive 80/777/EC provides supplementary requirement for the member states, concerning natural mineral water. European directives are adopted by each country and harmonized in every country’s law. All regulations, both in the United States and Europe, have to be respected by bottling companies during the process of producing, packaging, transporting, and selling bottled water. It is vital, for bottling companies, to develop and maintain HACCP programs, following the Codex Alimentarius Commission and FDA directives in the United States or the EN ISO 22000:2005 protocol in Europe. These quality control programs can ensure product safety and sustain public health during production and consumption of bottled water, while proving company’s respect to customers. Bottled water, sold both in the United States and in Europe, may be subjected to a number of treatment – disinfection methods. Absolute 1 mm filtration forces water to flow through filters to capture and remove particles larger than 1 mm in size, like Cryptosporidium. Ultraviolet irradiation, using 220–330 nm wavelengths, destroys bacteria, but endospore and viruses are not generally susceptible to this treatment. The use of ozone gas (ozonation), instead of chlorine, could act as an antimicrobial agent to disinfect water. Ozone leaves no residual taste or odor to the water. Reverse osmosis forces the water through semipermeable membrane, to remove constituents such as minerals with a molecular mass in excess of 150–250 Da. After the disinfection process, bottled water is usually distributed as a packaged food product, in glass or plastic containers from polyvinylchloride (PVC) or polyethylene terephtalate (PET), of various capacities (from 330 ml up to 20 l). The use of PET bottles for bottling water has become more popular due to the desirability of its physical and chemical properties, such as strength, transparency, lightweight, and toxicological safety. PET manufacturers declare that there is less than 1 ppm acetaldehyde in this material.

Bottled Water Microbiology All processes followed by bottling companies during production are disinfection, rather than sterilization processes, allowing a number of microbes, originated from source water to survive while eliminating pathogens. Bottled water as an end product rarely contains potential pathogens unless contamination occurs during production in the bottling plant, with the most likely sources of contamination being the equipment, bottles, and caps, exposure to air, and contact with working


personnel. The microbial quality of bottled water is usually expressed in terms of numbers of bacteria present in a given volume of water. EC Directive of 1980 prohibits the existence of total coliforms, Escherichia coli, Enterococcus spp., and Pseudomonas aeruginosa in 250 ml of any bottled water. Regarding the HPC standard of bottled water, EC sets an upper acceptable limit of 100 cfu ml 1 at 22 1C and 20 cfu ml 1 at 37 1C. Bottled water should be free from parasites, pathogenic microorganisms, and sulfite reducing anaerobes in any 50 ml sample. In contrast, the US standards include requirements for microbiological quality that are based on coliform detection levels, with HPC upper acceptable of 500 cfu ml 1 and a maximum level for coliforms 1 cfu per 100 ml. Additionally, the US legislation classifies both bottled water and bottled mineral water as bottled water. Bottled water originated from spring water sources usually contains little organic matter and bacterial flora consisting of Pseudomonas, Acinetobacter, Achromobacter, Aeromonas, and Flavobacterium species as well as other Gram-negative forms. Bottled water is a closed system, unlike tap water that flows through pipes. Once the bottle is filled and sealed, bottled water may remain on super market shelves or stored at home for weeks or months. Any bacteria that survive the disinfection processes may attach to the inside of the bottle and multiply, helped by any organic matter that is present in the water. Hence, water containing few organisms during bottling may show a bacterial logarithmic increase after a relatively short time. Bacterial growth follows the typical growth curve until all organic matter has been depleted. Additionally, in case bottled water is stored at room temperature, microbial concentrations of 104–105 cfu ml 1 can be reached in a few days. The problem may be further deteriorated in the case of plastic containers since plastic tends to be more permeable to external oxygen and extraneous vapors, whereas there is a possibility of nutrient release that could sustain bacterial growth. In the case of carbonated water, the low pH decreases or even prevents bacterial growth. However, uncarbonated bottled water is now considerably more popular than carbonated, being a substitute for tap water and often regarded as safer and healthier by consumers. Numerous studies in uncarbonated bottled water present an increase in microbes after 1–3 weeks of storage, with mainly Gram-negative nonfermentative rods (usually Pseudomonas spp., and related genera), originating from the water itself (autochthonous microflora) or because of contamination during the bottling process (allochthonous microflora). Many Pseudomonas spp., recovered from water are resistant to antimicrobial agents. Besides being a primary cause of disease, P. aeruginosa is often monitored as an indicator of other bacterial contaminants of fecal origin. In addition, there is a synergistic effect of P. aeruginosa on the survival of Salmonella


Heterotrophic Bacteria in Bottled Water

in water. The importance of Pseudomonades in bottled water is related to their potential as opportunistic pathogens (especially those species resistant to antibiotics) and their potential to contribute off-flavors or taints to water. Gram-positive bacteria have also been reported in bottled water, with the ability to form biofilms in pipes of bottling plants.

HPC Definition and Analysis HPC bacteria can be simply defined as microbes that require organic carbon for growth, and they include bacteria, yeasts, and moulds. They are widely used as indicators of drinking water quality and can be detected by propagation on nonselective media rich in nutrients to support the multiplication of the widest possible range of bacteria. Various HPC bacteria definitions have been reported by researchers in the past 20 years. Initially, the HPC term was introduced for facultative psychrotrophic Gram-negative rod-shaped bacteria, whereas later the autochthonous water bacteria were excluded. In the modern designation, the term HPC could include Gram-negative, nonfermentative, rod-shaped bacteria (e.g., Acintobacter, Flavobacter, Moraxella, and Pseudomonas), Gramnegative, fermentative, rod-shaped bacteria (e.g., Aeromonas, Enterobacteriaceae, and Vibrio), and Gram-positive bacteria (Bacillus, catalase-positive cocci, Corynebacterium, and Enterococcus). When assessing the public health impact of HPC, the taxonomy, virulence traits, and abundance of these bacteria have to be established. HPC bacteria in EC can be assessed at two recovery temperatures: 22 1C for 72 h and 37 1C for 24 h. Nitrocellulose membranes of 0.45 mm pore size and 47 mm diameter can be used for the filtration process, with readymade sterile ampoule microbiological media for the cultivation process. The 37 1C incubation temperature can be used as an indicator of fast-growing bacteria more likely to be related to pathogenic types, and the 20 1C incubation temperature can be used to enumerate characteristic water bacteria tend to develop slowly. Microbial techniques in the United States used for the enumeration of HPC bacteria have been reported by researchers: pour plate, spread plate, and membrane filtration. Pour Plate Method It is a simple, cheap, and easy method that can be used with nonselective media for HPC enumeration and with selective media for recovery and detection of specific target microorganisms. However, this method has three main drawbacks: melting agar temperature, small colonies formation, and small volume of sample being analyzed.

During analysis, the medium agar is melted from 43 to 46 1C, which causes an additional stress to bacteria, already suffering physiological stress in bottled water, thus reducing significantly bacteria recovery. The small colonies formation is due to the entrapment of bacteria in the agar medium and the creation of microaerobic environment, resulting in poor morphological characteristics that could assist the identification. Finally, the small volume of sample (1 ml) being analyzed limits the usefulness of the method, when analysis of a larger sample volume is needed. Spread Plate Method The main difference from the previous method is that the sample is distributed over the surface of the agar. Petri plates containing the agar medium have to be prepared ahead of time and checked for contamination before the analysis. The growth conditions in a spread plate method do not suffer because of an extra heat stress, as the colonies are on the surface exposed to aerobic conditions, resulting in higher bacterial counts than the pour plate method. However, the usability of this method is limited to the analysis of a maximum sample volume of 1 ml. Membrane Filtration Method This is the most flexible method for HPC determination, but more expensive compared to the previous two methods. It requires certain equipment, such as a vacuum pump and specific membranes with certified pore size, to entrap HPC bacteria. The great advantage of this method is the sample volume that can be analyzed, ranging from 1 ml to 10 l, depending on the water quality (membrane pores can be plugged or suspended material can interfere with bacterial growth). Thus, very low concentrations of bacteria can be detected. Depending on the needs of the analyst or the user organization, rich culture media such as plate count agar (PCA) or low-nutrient agar such as R2 A can be used at an incubation temperature of 3570.5 1C for 4872 h. Petri dishes are usually incubated from 24 to 72 h, at temperature ranging from 20 to 37 1C, and the HPC bacteria estimate is obtained by counting all bacterial colonies visible to the naked eye, under the specified conditions. However, the use of HPC criterion in evaluating bottled water quality is a contentious issue all over the world, mainly because of the variable degree of treatment needed each time a disinfection process is applied to produce bottled water, which depends on the quality of raw water sources. Limitations to identification of HPC species by commercial databases, such as API (bioMe´rieux’s microorganism identification test kits) galleries, can limit bacterial identification.

Heterotrophic Bacteria in Bottled Water

HPC Epidemiology The HPC bacteria of bottled water have been evaluated by several researchers in the past two decades, in United States, Europe, and Asia. A number of reports have shown that still (uncarbonated) mineral water has much higher bacterial counts (up to 105–106 cfu ml 1) compared to carbonated mineral water. Some bottled waters were found with fecal contamination, whereas some pathogens, such as Vibrio cholerae, Pseudomonas spp., and Aeromonas hydrophila, were reported, causing concern about public health. Although, the initial microbial quality of processed water is small, it can rapidly evolve to high numbers during storage. Bacterial multiplication may occur during 1–3 weeks after bottling, with bacterial counts reaching 104–105 cfu ml 1. Storage at ambient temperatures will aid the multiplication of contaminants that exist in bottled water. In the United States, several studies have been performed investigating the usefulness of noncarbonated bottled water for human consumption. One study investigated 23 different brands of noncarbonated bottled water that had undergone some form of purification treatment (ozone, carbon filter, micron filter, reverse osmosis, ion exchange, and steam distillation). Thirty percent of the samples examined were found to exhibit HPC bacteria counts more than 500 cfu ml 1, whereas Gramnegative bacteria, predominantly Pseuodomonas, were the most commonly recovered organism. Common bottled water HPC bacteria levels in United States are approximately 105 cfu ml 1, which with an average consumption of 8–14 l per week gives an estimate of 1.5–2.5 107 cfu per week intake from human organism. This amount is very low, consisting only up to 5% of the individual’s total bacteria intake from water, with food being the main source of bacteria ingestion in an average person. HPC bacteria do not represent a significant source of HPC in the average diet of consumers in the United States. Studies of bottled mineral water in Brazil revealed a P. aeruginosa contamination more than 50% in the samples examined (new and reused 20-l bottles). The number of samples with HPC over the maximum level legally permitted in Brazil (500 cfu ml 1) were 87% and 45% for reused and new bottles, respectively. According to this research, the bacteriological quality of municipal tap water was found superior to the quality of mineral bottled water of domestic origin. Bottled water studies in Australia, both domestic and foreign origin, have shown that species of Aeromonas were the predominant microflora in the majority (64%) of the mineral water examined. Pseudomonas mentocina was isolated from two foreign brands, indicating possible geographical specificity. Thirty percent of samples examined showed HPC bacteria levels above 104 cfu ml 1, enough to produce off-odors in mineral water.


Bottled water studies in the United Kingdom showed initial microbial numbers of up to 104 cfu ml 1, with an overall decrease in microbial level after 6 months of storage. A small portion of the bottled water showed no bacteria initially or after the 6 months storage period. This fact indicates the presence of toxic substances as suggested by some researchers. A significant interaction between age and bottled water brand was declared by this study as microbes in different bottled water brands were affected differently by aging. Ambient temperature was found to be critical, with more bacteria recovered at 25 1C storage temperature compared with 15 1C or 10 1C storage temperature. Gram-negative bacteria were found to overpass Gram-positive by 97.5% and 93.4%, after screening 700 samples initially and 300 samples after 6 months of storage, respectively. Gram-negative, oxidative bacteria were found to constitute more than 50% of the isolates on both sampling steps, whereas Gramnegative fermentative organisms were not identified at any stage. Gram-positive, catalase-positive, fermentative bacteria were found occasionally in some bottled water brands. This specific study suggested the use of surface culture techniques with low-nutrient agar media and longer incubation periods at lower temperatures to ensure that all viable bacteria in bottled water are recovered. In Italy, research was performed in bottled water by inoculating strains of P. aeruginosa, at a density of 102 cfu ml 1. The bottled water had low organic content and was packaged in reused returnable glass bottles. After 4–5 days, P. aeruginosa counts were increased by 3 log units, and these levels were maintained until 70–100 days after inoculation, after which a slow decrease began to appear. In Greece, studies performed in bottled water of domestic origin in a time period of 8 years identified species of Pseudomonas, Aeromonas, Pasteurella, Citrobacter, Flavobacterium, Providencia, and Enterococcus. Bottled water was sealed in PVC bottles, and samples were purchased from retail outlets, stored at approximately 20 1C. Pseudomonas aeruginosa was identified in 6% of samples, where HPC bacteria counts were found to be less than 20 cfu ml 1 in 82% (for HPC at 22 1C) and 86% (for HPC at 37 1C). Only 2% (for HPC at 22 1C) and 1% (for HPC at 37 1C) of samples were found with HPC levels greater than 1000 cfu ml 1. Other studies in Asia reported contamination with A. hydrophila and P. aeruginosa in specific bottled water brands, with a rapid increase in HPC levels to 104– 105 cfu ml 1 after storage at 25 1C. More than 50%, for both domestic and imported bottled water, were found with HPC higher than 200 cfu ml 1, and the remaining percentage consisted from HPC bacteria counts over 1000 cfu ml 1. Contamination during bottling and growth during storage are the main causes for HPC bacteria numbers, whereas autolysis of one organism providing


Heterotrophic Bacteria in Bottled Water

nutrients for the others could explain the rapid disappearance of E. coli in bottled water, within a few days of storage, as it was suggested by this specific study. Other studies, in specific Asian countries with no legislation on chemical and microbiological standard on bottled water, showed alarming results compared to HPC bacteria levels. Nearly 8% of bottled water of domestic brands were found contaminated with Pseudomonas spp. with 4% of the isolates P. aeruginosa. Staphylococcus epidermitis, Klebsiella pneumoniae, Enterobacter spp., and Acinetobacter lwoffi were also identified, although in the same research all bottled water of foreign origin were found free of Pseudomonas spp. Studies, by different researchers, showed a much higher rate and frequency of microbial contamination in bottled water packaged in 20 l compared to bottled water packaged in 1.5 l, with Acinetobacter species being the most frequently detected microbes.

HPC Pathogenesis and Antibiotic Resistance Many people think of bottled water as pure and tap water of lower quality, and in the case of gastrointestinal problems after a meal, they tend to suspect the food as the main cause. Even though bottled waterborne outbreaks are rare, individuals do not usually report an isolated incident. However, outbreaks have been reported due to V. cholerae in Portugal in 1974 and in Marianas Islands in 1994. Bottled water is often recommended for individuals in high-risk categories, such as immunocompromised patients, and often is shared among individuals. It has been suggested that there is a certain degree of risk due to the presence of opportunistic pathogens that are members of the HPC bacterial group. Hence, sterile water, frozen and thawed by the patient, may minimize the associated risk for illness. Pseudomonas aeruginosa is not commonly found in end products, and it is used more often as an indicator of contamination during the bottling process. However, some species of Pseudomonas have been implicated in nosocomial infection, as well as Acinetobacter spp., which are common inhabitants of some waters. Increasing drug resistance among HPC bacteria from bottled water is a fact open to question due to relatively few investigations into public health aspects of those products. As bottled water is produced without any heat processing, high HPC loads with possible opportunistic pathogens, especially multiple antibiotic resistance forms, can introduce public health problems in high-risk categories, such as immunocompromised individuals. Bacteria can quickly adapt to the presence of human-induced factors in the ecosystem, acquiring features that allow them to survive, such as resistance to antibiotics and xenobiotics. Water bottled in glass or plastic container can provide a suitable

ecosystem, which can be contaminated during production from working personnel. Source water, used for producing bottled water, can get contaminated with antibiotic-resistant strains due to bad agricultural practices (antimicrobial in farming) or due to discharging raw sewage into receiving waters. These bacteria could provide a suitable reservoir for spreading and multiplying resistance genes and their vectors. Species of Pseudomonas, regularly occurring in bottled water, have the ability to acquire and disseminate resistance genes, while being resistant to several antimicrobial agents, with susceptibility patterns similar to those of clinical strains. During patient antibiotic treatment, HPC bacteria with antibiotic resistance have the advantage against the normal flora, whereas this resistance can be transferred to susceptible organisms. The problem is further increased if the recipients are more virulent than the donor, as pathogenic bacteria. Studies investigating sensitivity of HPC bacteria isolated from bottled water have shown resistance to a large number of antibiotics: amikacin, ampicillin, chloramphenicol, ciprofloxacin, ceftazidime, streptomycin, tetracycline, whereas nearly half the isolates were found to possess multiple antibiotic resistance. There is increasing evidence that HPC bacteria might play an important role in the spread of plasmids due to the possibility to transfer resistance characteristics to nonresistant recipient cells via R-factor plasmid vectors. Drug-resistant HPC bacteria found in bottled water can act as a reservoir of resistance plasmids, which they can freely exchange with possible pathogens in the intestine. The production of bacteriocin-like substances from HPC bacteria could also provide the defending mechanism against competitors in bacterial ecosystems, favoring those microorganisms in colonizing bacterial habitats, by regulating population dynamics.

HPC Key Facts Bottled water originated from underground or municipal supply sources is rarely completely free of microorganisms. The majority of bottled water is subjected to some type of disinfection treatment, which does not include heat treatment. Hence, any HPC bacteria that survive during processing or introduced by contamination during bottling can infect the end product. HPC bacteria can survive and multiply in oligotrophic environments and finally enter to human recipients. HPC bacteria may include predominant species, such Pseudomonas, Vibrio, or Aeromonas, which have been implicated in waterborne outbreaks or denoted as vehicles of transferring antibiotic resistance. The careful use of bottled water by immunocompromised patients or individuals who share

Heterotrophic Bacteria in Bottled Water

bottled water can eliminate the potential for adverse effects, or close the entrance to potential or true pathogens. HPC microbial cells can attach to the solid surfaces of the bottled water containers, with PVC bottles found to promote adherence and colonization of bacteria. Glass bottles seem to be safer for bottling water, but with an increased cost for purchasing the glass containers and retaining a strict hygienic process throughout the bottling process (increased risk due to glass bottles returned to plant). Bottled water packaged in small PET containers (330 ml up to 1.5 l) seems to be safer than 20 l because of the single use of the small containers comparing to large ones that have to be returned to bottling plant for disinfection and refilling. The use of HACCP programs and GMP during processing, the correct transportation and storage at retail outlets at controlled temperatures, can reduce HPC bacteria levels and decrease the risk of HPC bacterial multiplication during storage. Uncarbonated bottled water exhibits greater variations and levels of HPC bacteria compared to carbonated bottled water that has lower pH, providing an obstacle to HPC bacteria multiplication. Under circumstances, HPC bacteria can increase the risk to public health, affect individuals by improper use of bottled water, or introduce health problems to immunocompromised patients. See also: Cyanobacterial Toxins in Fresh Waters, Giardia and Cryptosporidium: Occurrence in Water Supplies, Microbes and Water Quality in Developed Countries, Water Consumption and Implications for Exposure Assessment, Waterborne Disease Surveillance, Water-Related Diseases in the Developing World.

Further Reading Armas AB and Sutherland JP (1999) A survey of the microbiological quality of bottled water sold in the UK and changes occurring during storage. International Journal of Food Microbiology 48: 59--65. Bartam J, Cortuvo J, Exner M, Fricker C, and Glasmacher A (2004) Heterotrophic plate count measurement in drinking water safety management. Report of an expert meeting Geneva, 24–25 April 2002. International Journal of Food Microbiology 92: 241--247. Kokkinakis EN, Fragkiadakis GA, and Kokkinaki AN (2008) Monitoring microbial quality of bottled water as suggested by HACCP methodology. Food Control 19: 957--961.


Legnani P, Leoni E, Rapuano S, Turin D, and Valenti C (1999) Survival and growth of Pseudomonas aeruginosa in natural mineral water: A 5-year study. International Journal of Food Microbiology 53: 153--158. Messi P, Guerrieri E, and Bondi M (2005) Antibiotic resistance and antibacterial activity in heterotrophic bacteria of mineral water origin. Science of the Total Environment 346: 213--219. Mossel DAA and Struijk CB (2004) Assessment of the microbial integrity, sensu G.S. Wilson, of piped and bottled drinking water in the condition as ingested. International Journal of Food Microbiology 92: 375--390. Pavlov D, de Wet CME, Gradow WOK, and Ehlers MM (2004) Potentially pathogenic features of heterotrophic plate count bacteria isolated from treated and untreated drinking water. International Journal of Food Microbiology 92: 275--287. Ramalho R, Cunha J, Teixeira P, and Gibbs PA (2001) Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters. Journal of Microbiological Methods 44: 97--103. Ramalho R, Afonso A, Cunha J, Teixeira P, and Gibbs PA (2001) Survival characteristics of pathogens inoculated into bottled mineral water. Food Control 12: 311--316. Reasoner DJ (2004) Heterotrophic plate count methodology in the United States. International Journal of Food Microbiology 92: 307--315. Rosenberg FA (2003) The microbiology of bottled water. Clinical Microbiology Newsletter 25(6): 41--44. Sartory DP (2004) Heterotrophic plate count monitoring of treated drinking water in the UK: A useful operational tool. International Journal of Food Microbiology 92: 297--306. Stine SW, Pepper IL, and Gerba CP (2005) Contribution of drinking water to the weekly intake of heterotrophic bacteria from diet in the United States. Water Research 39: 257--263. Venieri D, Vantarakis A, Komninou G, and Papapetropoulou M (2006) Microbiological evaluation of bottled non-carbonated (‘‘still’’) water from domestic brands in Greece. International Journal of Food Microbiology 107: 68--72. Zamberlan da Silva MEZ, Santana RG, Guilhermetti M, et al. (2008) Comparison of the bacteriological quality of tap water and bottled mineral water. International Journal of Hygiene and Environmental Health.

Relevant Websites http://www.bottledwater.org/ International Bottled Water Association. http://eur-lex.europa.eu/ Official Journal of the European Communities. http://www.fda.gov/ US Food and Drug Administration. http://www.cfsan.fda.gov/ US Food and Drug Administration, Center for Food Safety and Applied Nutrition.