Role of IMs for Evaluation of DW Safety

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This entry is adapted from the peer-reviewed paper 10.3390/w15162964

Water safety and quality are essential for human development, well-being, and ecosystem health. Ensuring access to safe water is one of the most effective measures to promote health and reduce poverty. The contamination level of raw water directly influences the efficiency of drinking water (DW) treatment, subsequently impacting the quality of the produced DW. The microbiological quality assessment of DW and drinking water sources (DWSs) is based on the detection of indicator microorganisms (IMs). However, the relationship between IMs and pathogens has been questioned, as pathogens have been detected even in the absence of IMs, and vice versa.

drinking water drinking water sources indicator microorganisms coliforms Escherichia coli intestinal enterococci HPC

1. Introduction

To ensure that DW does not pose microbiological hazards to human health, it is essential to monitor its microbiological condition and verify the absence of pathogens. However, direct detection of all pathogens potentially present in DWSs and in DW is unfeasible for several reasons. The confirmed absence of a specific pathogen does not guarantee the absence of other pathogens or opportunistic microorganisms ^[1]. Additionally, there are not standardised detection techniques for all pathogens and the culture-dependent methods available for the direct detection of these microorganisms (i.e., multiple-tube fermentation technique, membrane filtration technique, standard bacterial cell culture methods, virus cultivation on cell cultures) are expensive and present certain disadvantages (e.g., labour-intensive procedures, extended lead times, and limited efficiency) ^[2]. Moreover, some of these methods exhibit low sensitivity, so they are unable to detect pathogens that, especially in cleaner water such as GW or DW, may be present sporadically and at low concentrations (e.g., human enteric viruses) ^[1]. Furthermore, culture-based methods are not able to detect the viable but non-cultivable (VBNC) state, which certain non-spore-forming bacteria may enter under stressful environmental conditions (e.g., E. coli, Vibrio cholerae, Helicobacter, Campylobacter) ^[2]. Similarly, these methods cannot identify viruses that are not cell-adapted and thus do not replicate on cell lines ^[3]. As a result, culture-based methods might underestimate the number of pathogens in a water sample, even risking declaring false negatives ^[4]^[5]. Subsequently, to address these challenges, molecular methods such as polymerase chain reaction (PCR), multiplex PCR (mPCR), real-time or quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), and droplet digital PCR (ddPCR) were developed and rapidly became widespread, as they are faster, more efficient, and more sensitive ^[4]^[5]. However, such methods can also detect the genetic material from dead or damaged microorganisms, complicating the interpretation of results in terms of viability and infectivity of the microorganisms and, thus, of the real risk to human health ^[6]. Due to this limitation, molecular techniques are not recognised as analysis methods in DW regulations. More recently, additional culture-independent methods have emerged (e.g., DNA microarrays, microbial source tracking MST, and next-generation sequencing NGS). Nevertheless, these methods are expensive and require highly specialised personnel, currently limiting their use primarily to research purposes ^[2]^[4].

Considering all these issues, the assessment of the microbiological safety of water is carried out using IMs. The ideal IM should meet the following characteristics: (i) present in water whenever pathogenic microorganisms are present, (ii) occur at concentrations reflecting the degree of water pollution, (iii) survive in water similarly to pathogens, (iv) not pathogenic, (v) applicable to any types of water, (vi) exhibit stable characteristics over time, (vii) not proliferate in water, and (vii) detectable through simple, rapid, and cost-effective methods ^[7]. Such an IM is not known to date, but over time, various complementary indicators providing different information have been proposed (Table 1). Indicators of faecal contamination are the most frequently mentioned in the literature. They are microorganisms typically abundant in the intestine of humans and other warm-blooded animals (e.g., E. coli, intestinal enterococci) and they are easily detectable in water. The detection of these IMs is considered predictive of the presence of pathogens transmitted through the faecal–oral route. Process indicators are sought in DW since they can demonstrate the effectiveness of water disinfection process (e.g., heterotrophic plate counts and total coliforms for chlorine disinfection). Lastly, index and model organisms are groups or species of microorganisms that indicate the presence and behaviour of pathogens, respectively (e.g., E. coli is considered an index organism for Salmonella, while F-RNA coliphages are considered model organisms for human enteric viruses) ^[6].

Table 1. Most widely used traditional and alternative indicator microorganisms (IMs), their main significance, and their use as surrogates for specific pathogens. The hyphen indicates the absence of utilisation as substitutes for specific pathogens.

IM	Significance	Surrogate for Pathogens
Total coliforms	Process indicators	-
Thermotolerant coliforms	Indicators of faecal contamination by humans and warm-blooded animals	-
Escherichia coli	Indicator of faecal contamination by humans and warm-blooded animals	Surrogate for bacterial enteric pathogens: pathogenic E. coli, Salmonella, Shigella
Intestinal enterococci	Indicators of faecal contamination by humans and warm-blooded animals	-
Heterotrophic plate count (HPC)	Process and water quality indicator	-
Clostridium perfringens and its spores	Indicator of remote faecal contamination by humans and warm-blooded animals	Surrogate for protozoan pathogens: Cryptosporidium, Giardia
Bacteroides spp.	Indicators of faecal contamination by humans and warm-blooded animals, they can be used to identify the source of faecal pollution	-
Coliphages	Indicators of faecal contamination by humans and warm-blooded animals	Surrogate for enteric viruses
Methanobrevibacter smithii	Indicator of human faecal contamination	-
Pepper mild mottle virus (PMMoV)	Indicator of human faecal contamination	Possible surrogate for enteric viruses

2. Total Coliforms

Coliforms belong to the Enterobacteriaceae family. They are Gram-negative, lactose-fermenting, non-spore-forming, oxidase-negative bacteria capable of aerobic and facultative anaerobic growth. These bacteria are present in the gastrointestinal tract of humans and animals, but they can also be isolated from environments without faecal contamination (e.g., water from food industries and biofilms within the distribution network). In addition, they survive in the environment by becoming part of the normal bacterial flora in freshwater of temperate and tropical environments. Hence, total coliforms are not specific indicators of faecal pollution, but they function as operational indicators that can provide information on the general conditions of the DW distribution system. They are mainly used as indicators of the efficiency of DW treatments, and their presence is related to the deterioration of DW quality, due for instance to the phenomenon of microbial regrowth ^[8].

3. Thermotolerant Coliforms

The thermotolerant coliforms, also known as faecal coliforms, differ from total coliforms in their ability to grow at high temperatures; indeed, they produce acid and gas from lactose at 44.5 ± 0.2 °C. The group of thermotolerant coliforms is represented by microorganisms that inhabit the gastrointestinal tract of animals, thus serving as indicators of faecal contamination by warm-blooded animals. They exhibit a survival pattern similar to that of pathogenic bacteria. E. coli is the most prevalent thermotolerant coliform found in faeces, and its presence is seldom detected in the absence of faecal pollution. Other faecal coliforms, such as Klebsiella, Citrobacter, and Enterobacter, can grow in the environment and may be present even in the absence of faecal contamination. Consequently, thermotolerant coliforms are considered more specific indicators of faecal contamination than total coliforms, but less than E. coli ^[1].

4. Escherichia coli

E. coli is a rod-shaped, Gram-negative, lactose-fermenting bacterium belonging to the Enterobacteriaceae family. This microorganism is a member of the thermotolerant coliforms group and can produce indole from tryptophan and β-glucuronidase. E. coli is the most prevalent commensal in the intestinal tract of warm-blooded animals, where it lives naturally, and, through excretion of faecal material, it can reach the environment. Its detection in water is easy, thanks to the availability of rapid, easy-to-use, sensitive, and specific methods of analysis. Since E. coli is predominantly present in faeces, it is a microorganism widely and historically used as indicator of faecal contamination and of the possible presence of bacterial enteric pathogens. The indication provided by the presence of this microorganism is of recent faecal contamination, which may result from pollution of DWSs with human or animal excreta, DW treatment ineffectiveness, or DW distribution system failures ^[9].

5. Intestinal Enterococci

Enterococci are Gram-positive, facultative anaerobic, chain-arranged, oxidase- and catalase-positive bacteria. They were included within the Streptococcus genus until 1984, when they were classified as members of the Enterococcus genus, which includes more than 50 species, such as E. faecalis and E. faecium. Intestinal enterococci grow at the optimum temperature of 35 °C and live as commensals in the gastrointestinal tract of humans and other warm-blooded animals. Enterococci, along with E. coli, represent the most used indicators of faecal contamination because of their abundant presence in faeces; they are generally more abundant in faecal matter of animal rather than human origin. Enterococci have developed strategies of resistance to adverse environmental conditions; in fact, they are able to grow in environments with NaCl concentrations of 6.5%, at temperatures ranging from 10 to 45 °C, and at pH values of 9.6 ^[10]. They also exhibit increased resistance to dehydrating conditions and to chlorination, and they can survive for long amounts of time in the outdoor environment. These characteristics of resistance can be attributed to the thicker cellular wall typical of Gram-positive bacteria and their ability to form layers of biofilm in the DW distribution network, contributing to its deterioration and acting as a reservoir for other microorganisms. These characteristics make enterococci potential indicators of less recent contamination than E. coli and other coliforms, as well as of DW disinfection treatment efficiency. Although intestinal enterococci are commensal bacteria of humans and animals, some species can act as opportunistic pathogens, causing nosocomial infections because of their potential virulence factors and antibiotic resistance genes ^[8].

6. Heterotrophic Plate Count (HPC)

HPC, also known as total plate counts or colony counts, refer to the enumeration of heterotrophic microorganisms capable of growing on non-selective solid culture media without inhibitory or selective agents, and under defined cultivation conditions. HPC communities encompass a wide range of ubiquitous heterotrophic bacteria that naturally exist in various environmental matrices, as they use organic nutrients for growth. The effectiveness of DW treatments and the integrity of DW distribution systems are evaluated by the count of colonies grown at 22 °C, which represent the presence of microorganisms that are naturally found in the environment and, therefore, that are not related to faecal pollution. On the other hand, colonies grown at 37 °C indicate the presence of microorganisms originating from humans or animals ^[11]. Since the number of heterotrophs should be reduced by DW treatments, elevated colony counts or substantial increases in their numbers indicate DW quality deterioration due to treatment deficiencies, loss of disinfection residual, biofilm formation along the distribution network, or stagnant water conditions ^[12].

7. Clostridium perfringens and Its Spores

Microorganisms belonging to the Clostridium genus are spore-forming, anaerobic, and sulphite-reducing bacteria. Considering their ability to produce spores as forms of survival in adverse environmental conditions, clostridia possess high stability in water and high resistance to environmental stresses and to disinfection processes. C. perfringens is the representative species of this genus; it is part of the intestinal microbiota of humans and other warm-blooded animals and its origin is entirely faecal. C. perfringens and its spores are widely distributed in sewage and do not multiply in water environments. For these reasons, C. perfringens spores are used as indicators of remote or past faecal pollution and as surrogates for pathogens resistant to water treatment processes. Therefore, the presence of C. perfringens spores in DW could indicate contamination by protozoan cysts/oocysts, highlighting deficiencies in treatment and disinfection processes or possible recontamination of the distribution network ^[13].

8. Bacteroides spp.

Microorganisms belonging to the Bacteroides genus are Gram-negative, non-spore-forming anaerobes. They exclusively inhabit the intestinal tract of warm-blooded animals, where they account for a 1000-fold greater portion than coliforms and show lower survival rates in aquatic environments than coliforms. The use of these microorganisms as indicators of faecal pollution has shown limitations due to the difficulty of cultivation with traditional bacteriological methods. However, the advances in molecular methods allow for overcoming these problems, resulting in rapid detection and identification of these bacteria ^[14]. In addition, certain species of the Bacteroides genus are highly host-specific, which means they are found in a particular host organism and not in another. PCR methods have been developed to detect the genetic marker sequences specific for both Bacteroides and the host species (e.g., human- and bovine-specific Bacteroides 16S rRNA genetic markers), making it possible to distinguish and identify the source of faecal pollution in water (e.g., human or bovine) ^[15].

9. Coliphages

Bacteriophages or phages are viruses that exclusively use bacteria as hosts for their replication. Coliphages are bacteriophages that infect E. coli and closely related coliform bacteria, so they are found in the faeces of humans and other warm-blooded animals. Coliphages share numerous characteristics with human viruses, particularly in terms of composition, morphology, structure, and replication mechanism. As a result, they have been proposed as useful models or surrogates for evaluating the behaviour and resistance to treatment and disinfection processes of enteric viruses in aquatic environments ^[16]^[17]. Coliphages used in water quality assessment are divided into two main groups: somatic coliphages and F-specific coliphages. Somatic coliphages belong to the Myoviridae, Siphoviridae, Podoviridae, and Microviridae families and exhibit a wide spectrum of morphologies. They infect E. coli by attaching to receptors permanently located on the cell wall. They generally replicate within the gastrointestinal tract of warm-blooded animals but can also replicate in some aquatic environments. Because of their mode of replication and host specificity, somatic coliphages are excreted by most humans and animals and they are found in aquatic environments at higher concentrations than F-specific coliphages. F-specific coliphages, also called sex coliphages or male-specific coliphages, initiate infection by attaching to sex pili (F pili) produced by E. coli cells containing the F plasmid responsible for bacterial conjugation. Since F pili are produced only during the logarithmic growth phase at temperatures exceeding 30 °C, F-specific coliphages are unlikely to replicate in environments other than the gut of warm-blooded animals. F-RNA coliphages are a subgroup of F-specific coliphages containing a single-stranded RNA genome and belong to the Leviviridae family, comprising the two genera Levivirus and Allolevirus. F-RNA coliphages are excreted by a variable and generally lower percentage of humans and animals than somatic coliphages ^[18].

The presence of coliphages in DW indicates faecal pollution and the potential presence of enteric viruses. It can also highlight deficiencies in water treatment, as coliphages exhibit resistance to disinfection processes such as chlorination and UV radiation ^[19].

10. Methanobrevibacter smithii

Microorganisms belonging to the Methanobrevibacter genus are members of the Methanobacteriales order and belong to the Archaea domain. Some species of the Methanobrevibacter genus live in the digestive tract of animals, especially ruminants (e.g., M. acididurans and M. wolinii in sheep, M. gottschalkii and M. thaueri in horses and pigs, and M. ruminantium in ruminants in general), where they enhance the digestion of cellulose thanks to their metabolism of methanogenesis. The most prevalent and abundant species in the human intestinal microbiota, detected in more than 50% of the adult population, is M. smithii, which colonizes the cecum to rectum tract and employs H₂ or formate to reduce CO₂. Currently, M. smithii has only been detected in human faeces and not in the faeces of other animals. Owing to its host specificity and high abundance in the human gut, M. smithii may be a useful human-specific marker of faecal pollution in aquatic environments ^[20]^[21].

11. Pepper Mild Mottle Virus

Pepper mild mottle virus (PMMoV) is a plant-pathogenic (Capsicum spp.) positive-sense, single-stranded RNA virus that belongs to the family Virgoviridae and the genus Tobamovirus. PMMoV virions enter the human system through food, particularly via the consumption of peppers and processed products such as curry and hot sauce. Subsequently, these virions are excreted with faeces into the environment. Virions are extremely stable, withstanding standard food processing methods and various environmental conditions, and retaining their infectivity for plants after passage through the human gut, within which they do not replicate ^[9]. PMMoV has been identified as the most abundant virus excreted in the faeces of a wide percentage of healthy adults (10⁵–10¹⁰ copies/g of faeces) and has been detected at high concentrations in raw sewage throughout the world. In addition to wastewater, PMMoV has also been identified in DWSs and irrigation water and appears to be more persistent in aquatic environments than other viruses ^[22]. Despite being widespread in human faeces, PMMoV has rarely been detected in animal faecal samples (e.g., those from chickens, seagulls, geese, and cows) and at 3–4 log lower concentrations than in human faeces. For all these characteristics, PMMoV has recently been proposed as a potential viral indicator of human faecal pollution in aquatic environments and to evaluate the efficiency of DW treatment processes. However, further studies are needed to assess whether different food preferences and ecological distribution of host plants may influence the presence and abundance of PMMoV in different geographical regions ^[23].

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