Microbial Indicators and Their Use for Monitoring Drinking Water Quality—A Review

: An increase in the incidence of water-borne human diseases, such as diarrhea and emesis, has occurred due to drinking polluted water. These water-borne diseases can lead to death, if correct treatment is not provided. Assuring that drinking water quality is safe has been a crucial challenge for public health. Water contamination with pathogenic microorganisms represents a seriously increased threat to human health. Currently, di ﬀ erent microorganisms are being used as the primary indicator to assess water quality total coliform and Escherichia coli ( E. coli ) being the most common. However, increasing the occurrence of water-borne illness from sources deemed safe by the microbial standard criteria has raised the question—are these microbial indicators reliable and sensitive enough to ensure water quality? Currently, other microorganisms including bacteria, enteric virus, and protozoa are being tested and used in di ﬀ erent countries as alternative indicators to monitor water quality. It is necessary to study the diverse water quality indicator systems used throughout the world and their e ﬃ cacy with the present water quality. Although water quality standards suggest adding pathogenic microorganisms such as enteric virus as an indicator, China only uses pathogenic E. coli , protozoa. Pin-pointing the shortage of the current water quality indicator system in China is crucial in order to propose changes in future water quality indicator systems.


Introduction
Water is an indispensable resource that humans need to live by. Unfortunately, water is also an efficient medium in the transmission of diseases. Drinking water safety is a considerable public health concern. It is reported that 4.6% of global disability-adjusted life-years (DALYs) and 3.3% of global deaths is related to water quality [1]. Millions of people die from waterborne diseases every year all over the world [2,3]. With the growth of the economy, the water contamination problem is becoming progressively more serious, consequently, influencing the quality of drinking water. According to a research, 50% of global diseases are caused by contaminated drinking water [4]. This contamination can cause more than 50 serious illnesses including digestive disease, infectious disease, skin disease, cancer and so on. In the USA, 32 drinking water-related outbreaks were reported between 2011 and 2012, accounting for at least 431 cases of illness, 102 hospitalizations and 14 deaths [5]. Almost one third of the global population is living in developing South Asia where people are unaware of

Water Quality Indicator Systems and Criteria for Selection
Currently, there are a quite a few different types of drinking water quality standards being used in many individual countries. The three are most common suggested as global standards include a) USEPA National Drinking Water Criterion, b) EC Drinking Water Quality Directive and c) WHO Drinking Water Quality Criterion, which have served as a foundation for other national water quality standards.
In 2011, WHO issued a fourth Drinking Water Quality Standard, which includes the 28 microbiological indicators that are acknowledged waterborne pathogens. It contains 12 kinds of bacteria, 8 kinds of viruses, 6 kinds of protozoa and 2 kinds of parasites [22]. The presently used USEPA National Drinking Water Quality Standard, which was issued in 2009, is divided into two parts: the National Primary Drinking Water Regulation (NPDWRs) and National Secondary Drinking Water Regulation (NSDWRs). The NPDWRs belong to the compulsory standard for the public water supply system, with a total of seven microbial indicators used. In contrast, The EU Drinking Water Directive 98/83/EC, passed in 1998, contains only two microbial indicators. It is necessary to urge the standard to raise the requirements regarding the frequency of the indicators. For instance, several easily detectable indicators, such as E. coli, need to be tested more frequently and the full-scale detection needs to be done once these indicators exceed the standard [23].
This paper also conducted an in-depth analysis and comparison of water quality indicator systems from selected parts of world, specifically those used in Asian countries, European countries, the USA and Africa.

Water Quality Indicator Systems in China
The relationship between national water environment quality standards and human health has always posed a serious concern in China. Drinking water quality standards are standards that ensure the safety of drinking water and provide a basis for the department of health to monitor drinking water. The principles behind the Drinking Water Quality Standard in China are pathogenic microorganisms are not allowed in water, chemical substances and radioactive substances in water cannot pose harm to human health and good sensory properties of water.
In China, Shanghai was one of the first cities to establish local drinking water management system and the government issued Drinking Water Standards in 1950. Following the establishment of Drinking Water Standards, the Ministry of Health drew up a drinking water provisional act in 1955 and put it into effect in twelve big cities. Then in 1959, the provisional act was renamed as The Drinking Water Quality Standard by the Ministry of Health, which contained two bacterial indicators: aerobic bacterial count and total coliform [24]. With increasing incidence of illness caused by protozoa, Giardia and Cryptosporidium were then added to the standard in 2006. The specific indicators and limit values are shown in Table 1. The indicator aerobic plate count and total coliform are used to indicate microbial contamination. The former presents an overall microorganism contamination in water, the latter present the overall pathogenic microorganism contamination. However, some pathogenic microorganisms such as enteric viruses are more stable during chlorination, which can be a potential risk to harm public health. Therefore, enteric viruses are detected as an alternative indicator to assess water quality by some studies in China. For instance, Allmann Erin et al. tested for the presence of enteric viruses in recreational water in Wuhan using methods established at the University of Hawaii [25]. The study confirmed the resistance of enteric viruses to chlorination by detecting its presence in a previously approved recreational water source [25]. In addition, Huiling Chen et al. used the real-time RT-PCR method to detect enteric viruses in Shenzhen and concluded that it is necessary to reinforce continuous monitoring of enteric virus [26]. However, due to a number of reasons, enteric viruses, still, have not been used as a water quality indicator in China.

Indicators in Japan
The first drinking water quality standard in Japan was established in July 1955 by the Japanese Ministry of Health. This drinking water standard referred to the WHO Drinking Water Directives and was amended twice after being established. In 1993, Japan adopted general bacteria and coliform bacteria as microbial indicators. General bacteria cannot exceed ≥ 100 per mL and coliform bacteria cannot be detected in drinking water [27]. The new revision of water quality criteria was carried out in April 1st, 2015. The new microbiological indicators added to the revised standard were similar to those used in China. The revised standard states that total coliform, thermotolerant coliform bacteria, and Escherichia coli cannot be detected in water, and general bacteria cannot exceed ≥ 100 per mL [28].

Indicators in Singapore
Singapore is a tropical island nation located on the southernmost point of the Malay Peninsula in Southeast Asia [29]. Singapore is a nation without groundwater and its sources of water mainly depend on the four factors: desalination of sea water, new water, reservoir water and water supply from other countries such as Malaysia. The role of reservoir water is quite important, it not only provides a source of drinking water, but it also serves a recreational purpose. Therefore, the local government has paid more attention to the management of reservoir water sources and monitoring water quality. Singapore has been using the water quality indicator standards issued by the WHO for drinking water. The guidelines were drafted in 1965 by the WHO, which then issued a fourth guideline in 1986. A fifth guideline was issued during International Water Week in Singapore in July of 2011 [30]. The first and second guidelines, contained only two microbial indicator systems. When issuing the third guideline; however, the WHO began to pay more attention to the large variety of microbiological indicators, and added 25 new waterborne pathogen indicators. Up until 2011, the WHO has clearly suggested the risk of waterborne illnesses caused by polluted water, and listed microorganism causing the illness in the first place. Therefore, the latest guideline issued in 2011 contained 28 waterborne pathogen indicators, including 12 bacteria and 8 enteric viruses.

Indicators in Malaysia
In Malaysia, the main water sources are rivers and streams, which largely depend on rainfall [31]. The establishment of the drinking water quality standard in Malaysia was dependent on the standards recommended by the WHO and by Australia. According to the standard issued in 1990, the microbial indicators in Malaysia contained seven types. Total coliform and enteric viruses were chosen from the WHO's Drinking Water Quality Standards issued in 1984. Two methods are currently used to detect the total coliform: most probable number (MPN) and the membrane filtration method. These two methods have different indicator limitation requirements. The advantage of this standard is the determination of test frequency according to different water sources (Table 2).

Indicators in European Countries
European Community (presently called "European Union"), which consisted of a total of 28 countries such as France, Germany and other countries, issued standards concerning the quality of surface water with the intention for the abstraction of drinking water in the Member States (75/440/EEC) in June 1975. Currently, two important instructions for water protection are (1) The European Union Water Quality Directive (98/83/EC) [32] and (2) The Water Framework Directive (2006/60/EC) [33,34]. In the first instruction, microbiological indicators for drinking water include E. coli and Enterococci, according to the directive, water is considered safe for consumption if no microorganisms are detected in 1 mL [33,34]. The directive was suggested for every member country, and each member country can amend indicators based on their own situation. The majority of countries in Europe have adopted the above instructions; but there are several countries, such as the United Kingdom, France and Germany who have established standards based on European Union Water Quality Instructions (EUWOI). Russia, however, has established their own standards different from EUWOI and WHO.
The UK possesses a developed economy and the most abundant water sources, predominantly rivers, among the EU. In 1996, The Surface Water (Abstraction for Drinking Water; Classification) Regulations of 1996 was issued in England and Wales. The regulations required bacterial indicators stipulated, like Enterococci and E. coli, to be 0 per 1 mL in faucet water and coliform and E. coli to be 0 per 1 mL in reservoir water and water from water treatment plants [35]. This standard was consistent with European Union Water Quality Directive. In 2000, Water Supply (Water Quality) Regulations 2000 required surface water sources for drinking water to be the same as the 1996 standard. Three organizations are in charge of the system for monitoring water quality: The Office of Water Services, Drinking Water Inspectorate and Consumer Council for Water.
Predominately used Drinking Water Quality Criterion (95-368) in France refers to EU 80/778/EC, with the addition of three new amendments made in 1990, 1991 and 1995. Most index values used were the European highest tolerant level, especially the comprehensive microbiological standards. The standard contained seven microbiological pathogens, five bacteria indicators and two viruses. For bacterial indicators, both E. coli and fecal enterococci was required to be 0 per 100 mL, sulfite-reducing Clostridia was required to be < 1/20 mL, Salmonella was required to be 0/5 L and Staphylococcus was 0/100 mL. For viral requirements, a phage was to be 0/50 mL and no detection of enteric viruses per 10 L [36].
The majority of rivers in Germany, a country located in central Europe, are transboundary waters. Even though water shortage is not a problem plaguing Germany, water pollution is a great concern. In the 1970s, Germany experienced rapid economic development. Along with economic development came serious environmental pollution. Therefore, rather than participating in the draft of policy and rules in the EU, Germany established several standards of its own for the international rivers. Germany began enforcing France's Drinking Water Law on May 22, 1986. What is more, Germany's standard in 2000 clearly required drinking water to meet the following requirements: (1) it must be without pathogens, (2) without risk to human bodies and (3) without the majority of microorganisms. The standard for E. coli was 0 per 100 mL for pipeline water and 100/1 mL for treated water [37].
Russia's capital city Moscow holds three quarters of the Russian population. Drinking water in Russia mainly comes from central water supply. Biological contamination reached 27% in Evenk Autonomous Okrug, an urban-type settlement in central Russia, while other areas' biological contamination were relatively lower (2.5%-12%). The pollution sources mainly result from general bacteria (17.5%) and E. coli (12.5%) [38]. Russia's drinking water standards were established by their ministry of health with special characteristics. Since their standard level is similar to the international level, the Russian government formulated a national water standard: The Drinking Water Standard 2874-82, and a health standard called Standard 2.1.4.599-96 for new water plants set up after 1998. The microbial indicators of the two standards are shown in Table 3.

Indicators in the United States of America (USA)
The United States established the Environmental Protection Agency (EPA) on December 2nd 1970 in order to protect humans from risks caused by environmental pollution. The EPA developed documents and criteria of water quality to make sure water is safe for human use. The first water quality criterion in the United States was proposed in 1968 by the National Technical Advisory Committee (NTAC) of the department of federal government. Several microbial indicators were used for measuring recreational waters including total coliform, fecal coliform, E. coli, and enterococci [39]. Total coliforms and fecal coliforms were proposed by the US Environmental Protection Agency (EPA) in 1976. At that time, the EPA recommended the standards for recreational water in the Water Quality Criteria, which states "Based on a minimum of five samples taken over a 30-day period, the fecal coliform bacteria level should not exceed a log mean of 200 per 100 mL, nor should be more than 10 percent of the total samples taken during any 30-day period exceed 400 per 100 mL". This criterion, which was first proposed by the National Technical Advisory Committee (NTAC) to the Federal Water Pollution Control Administration in 1968, was adopted by nearly 95% of the states and territories in the United States. Fecal coliform appearing in drinking water and/or swimming sites indicates that the water has been polluted by human or animal feces. The coliform bacteria (total coliform and fecal coliform) do not fully reflect the occurrence of pathogens in disinfected wastewater due to their relatively high susceptibility to chemical disinfection and failure to correlate with protozoan parasites and enteric viruses [40]. In addition, coliforms are usually considered as irresponsible indicators of fecal contamination because they are able to grow in the environment [41]. Therefore, alternative indicators have been advised for evaluation of drinking water and recreational water including enteric virus, Enterococcus, Clostridium perfringens and coliphage [40]. A study by Dufour that was conducted at three marine beaches and two freshwater beaches between 1972 and 1982 revealed a direct linear relationship between swimming-associated gastroenteritis and the concentration of two bacterial indicators, enterococci and/or E. coli in water, but no statistical relation with fecal coliform concentration. Due to the strong relationship between the concentration of E. coli and/or enterococci and the rate of gastrointestinal illness, the US EPA amended the use of E. coli and enterococci as indicators of fecal pollution in state recreational water-quality criteria in 1986. The 1986 Recreational Water Quality Criteria (RWQC) set the water quality criteria of enterococci at geometric means of 33 and 35 CFU/100 mL [42]. For about a century, the fecal indicator bacteria (FIB) had been used to assess water quality to protect human health through monitoring fecal indicators. However, Ostrolenk et al. proposed in the year of 1947 that the enterococci may be a more suitable FIB than E. coli [43]. This proposition was further confirmed by studies conducted in the 1970s [43,44]. Currently, the only FIB species recommended by the US EPA is enterococci for swimming-related water, since it shows a stronger correlation with human health effects than other FIBs, such as fecal coliform and E. coli [44,45]. Other pathogenic indicators, like Clostridium perfringens, have been considered as potential indicators of water pollution in an aquatic environment [46]. Currently, water quality is monitored using National Primary Drinking Water Regulations (NPDWR) established in 2001, which is the compulsory standard. The microbial indicators in the standard are shown in Table 4. The standard includes Cryptosporidium, Giardia, Legionella and enteric virus, which are not common in other national standard listed. The standard also includes turbidity as a microbial indicator because high turbidity levels are usually related to the high levels of other pathogenic microorganism such as virus and bacteria [47]. The USEPA recommends several standards of E. coli and enterococci for fresh water [42]. In October 2000, US Congress required states to adopt the USEPA criteria and establish a monitoring program [48]. However, ecology and physiology, survivability of treatment and varying number of bacteria indicators relationships to pathogens, caused some states to add monitoring parameters specific to their local concerns rather than developing the site-specific criteria for themselves. Hawaii, Southern California, Great Lakes states and Southern Florida are four different regions containing popular beach destinations in the USA [44]. In 2012 the Recreational Water Quality Criteria (RWQC) compared these states with each other and discussed regional differences in climate, water condition, distribution of animals and plants, frequency of rain or dry and even difference between coastal water and inland water [42]. Since 1958, California used a single total coliform (TC) standard as the local recreational water quality standard, however, a change in July 1999 suggested a standard requiring measurement of three indicator organisms: total coliform, fecal coliform and E. coli [49]. Hawaiian streams are classified as recreational waters and therefore require < 200 fecal coliform /100 mL, these water quality criteria were established in 1982. Since FIB generally occurs in ambient environment and has high concentrations in soil and water, C. perfringens has been used as a second recreational water quality indicator to provide better water monitoring in Hawaii.
The concern now in the US focuses on the study of enteric virus and other indicators that can reflect fecal contamination. US indicators rely solely on bacterial indicators such as total coliforms, fecal coliforms and enterococci for inferring water microbiological quality, however, bacteria indicators do not always indicate the quality of water in a situation where the water meets the bacterial standards but contains a low number of viruses.

Indicators in the Canada
Canada is a country in North America. It is north of the United States. Canadian drinking water supplies are generally of excellent quality. However, water in nature is never "pure". It picks up bits and pieces of everything it comes into contact with, including minerals, silt, vegetation, fertilizers and agricultural run-off. While most of these substances are harmless, some may pose a health risk. To address this risk, Health Canada works with the provincial and territorial governments to develop guidelines that set out the maximum acceptable concentrations of these substances in drinking water. These drinking water guidelines are designed to protect the health of the most vulnerable members of society, such as children and the elderly. The guidelines set out the basic parameters that every water system should strive to achieve in order to provide the cleanest, safest and most reliable drinking water possible. The most significant risks to people's health from drinking water come from microscopic organisms such as disease-causing bacteria, protozoa and viruses [50]. The guidelines that relate to these microorganisms are stringent because the associated health effects can be quite severe. They can also affect health over the long-term. The microbial indicators are shown in Table 5.

Indicators in Australia
Australia is a developed country belonging to Oceania with water shortage concerns making water sources very important to the public. Therefore, in terms of the safety of drinking water, Australia established comparatively perfect indicator standards based on the three authoritative standards spelt out by the WHO, EU and USEPA. The nation managed its water sources through cities and towns. The currently used drinking water standard was revised in 1996. It is worth pointing out that the standard not only contained microbiological indicators, but also listed irregular testing microbiological indicators. There were a total of 22 microbiological indicators including bacteria, virus, protozoa, toxic algae, E. coli and total coliform all in which cannot be detected per 100 mL of water, including two microbiological indicators (Escherichia coli and total coliform), and twenty irregular testing microbiological indicators, which contain several microbiological indicators that can not be tested in water, otherwise it is necessary to adopt corresponding measures for the government and several microbiological items that have not been used as indicators due to inadequate data. Furthermore, the regulation requires microbial test to be performed once a week for surface water and once for 2 weeks for groundwater. Enteric virus: adenovirus, enterovirus, hepatitis virus, norovirus and rotavirus, all can cause gastroenteritis through contaminated drinking water [51]. Although those indicators are not given limiting values, they are listed in the standard to protect public health. Meanwhile, there were several microbiological species, which were not used as indicator due to insufficient data such as Legionella and Pseudomonas. Drinking water standards in Australia have relatively wide coverage, concerning the most microbiological indicators that may pose risk of human health.

Indicators in New Zealand
New Zealand is a country of two large islands (called the North and South Islands) and many much smaller islands in the South-Western Pacific Ocean. The National Environmental Standard (NES) for Sources of Human Drinking Water came into force in June 2008. The Drinking-Water Standards for New Zealand (DWSNZ) are an important resource for monitoring whether water is safe to drink. The indicator organism Escherichia coli (E. coli) is used in the DWSNZ to assess the bacterial quality of water. The bacterial quality of treated water is satisfactory if the E. coli concentration is less than 1 organism per 100 mL. When E. coli is detected in water it shows that the water has been in contact with feces: this means that pathogens may also be present [52]. There is too little information available on which to base maximum acceptable values for a viral indicator or individual viruses. A virus suitable to act as a viral indicator (similar to E. coli for bacteria) has yet to be found.

Indicators in Africa
Currently, Africa projects demographic changes, which will lead to fast movement in population growth and urbanization. It is predicted that 57.7% of all Africans will be living in urban areas by 2050. The infrastructure in low-income countries does not follow the pace of demographic change and urbanization, which will increase the risk of water shortage and water contamination. Nearly half of population in Africa cannot get clean drinking water and two thirds of the population lacked the infrastructure for the management of feces. Fecal coliform has been the only indicator in the water quality criteria (WQC) in South Africa since 1984: microbial levels cannot exceed 100 CFU per 100 mL for direct contact recreational water and must be < 15 per 100 mL for drinking water. Water quality on the African continent is a challenge because it is unknown for larger parts due to very little measured data. Due to the rapidly increased population in Africa, water sources supply and public health service is scarce, with economic depression and natural hazard, leading to unsafe drinking water. In addition, there is no water quality criteria established for the whole African countries. Therefore, future concerns should focus on the establishment of water quality indicator for public health and introduction of facilities for fecal treatment.

Present Challenges with the Current Indicator System
Currently, drinking water standards issued by the WHO, EC and USEPA are the most advanced water quality standards on an international level and are the basis of standards for most countries around the world. Among these three standards, microbiological indicators contain two, five and seven items, respectively. The WHO listed the microbiological indicators as the important tool to monitor the safety of water quality. Microbiological pathogens are harmful to public health because they make people sick when exposed to contaminated water. This requires one to set up effective protection measures in the process of water source choosing, cleaning water methods, disinfecting and water supply. Current water quality monitoring systems is largely dependent on bacterial indicators, although there are other feasible methods adopted by several countries for detecting microbial pathogens. Bacteria such as E. coli, which represents fecal contamination, is an appreciated indicator to monitoring water quality. However, other pathogenic microorganisms, such as enteric viruses, is currently used in France, but have not yet accepted as alternative indicators for water quality monitoring in the majority of countries. Different countries have their own standards in terms of microbiological indicators. In the USA, drinking water standards-National Drinking Water Criteria by US EPA are relatively perfect, the enteric virus has not yet been included as an indicator for a variety of reasons including low concentration, infective nature, difficult detection and more costly for detection. Furthermore, epidemiological studies related to enteric virus resulting in waterborne diseases are short of data, which is a big concern holding current systems back from adopting enteric virus as alternative indicator. In China, studies about the enteric virus in water and studies involving bacterial indicators reflecting fecal contamination are very limited. One of the main challenges in adding water quality indicators is the variety of bacteria that can be used. For example, Clostridium perfringens is used in Hawaii as an alternative indicator because of its relationship with a waterborne illness in tropical areas. It is necessary to seek alternatives for monitoring water quality in different areas in China. Meanwhile, the limit value for bacterial indicator in the current standard are relatively more relaxed than countries such as Russia, therefore, more studies need to be done to analyze if the standard level used in today is effective to indicate today's water quality and fecal contamination situation. It is reported that the number of cases of waterborne illness in China remains high and 123 students were infected hepatitis A caused by polluted drinking water in the Sichuan province in 2004.

The Need for a New Indicator System-Future Indicator Systems in China
Concern about the drinking water safety is increasing in China due to potential exposure to microorganisms. To increase water quality, it indicates that China needs to establish a strict and more complete indicator system and strongly enforce water quality standards. The drinking water quality in China remained stagnant for about 11 years until recently. The increasing economic growth has subsequently caused water quality to decrease. The global concern for water quality is focused on microbiological indicators, therefore, it is a better way for future indicator systems to do the same by considering all possible bacterial indicators, considering enteric virus and protozoa as water quality indicators, and lastly, reevaluating the indicator limit value associated with the present water quality situation.

Summary
This article reviews the information about the drinking water quality indicator systems in different parts of the world. Water quality security has been a subject of great importance across the public health field. Governments and policymakers have been highly aware of the role water plays in the transmission of diseases. The established process in the WHO Drinking Water Quality Standard is strict, containing a number of parameters and the microbiological indicators total coliform and E. coli. The WHO Drinking Water Quality Standards serves as a foundation for several different quality standards around the world such as standards in the Philippines and Thailand. Further, the WHO's standards have been fully adopted by many large cities such as Hong Kong and Singapore. The EU Drinking Water Quality Directive contains a total of five bacterial indicators, however, some European countries, like France, listed enteric virus as an indicator for monitoring water quality. The USEPA Drinking Water Quality relies solely on microbiological indicators, listing a total of seven indicators, including the bacterial indicators fecal coliform and E. coli; and protozoa such as cryptozoite and virus. According to epidemiological data, some protozoa such as Giardia and cryptozoite are the pathogens most associated with waterborne illness. Due to this association the USA, the UK and Australia have added both pathogens to their standards. For China to maintain its water quality, it is necessary for the country to expand their microbiological indicators by considering adding other bacteria, enteric virus and protozoa as alternative indicators in their water quality standards.