Bacteriological Perspective of Water Quality

Abstract

Water quality can be defined as a measurement of a water’s appropriateness for a specific purpose based on biological, chemical, and physical qualities. Water pollution caused by microorganisms is one of the most serious threats to the aquatic ecosystem around the world. The bacterial concentration in an aquatic environment is raised by anthropogenic activities and industrial-agricultural pollutants. Coliform bacteria have long been used as an indicator organism for microbial pollution of water, which has contributed to potential health risks. Escherichia coli is the coliform that indicates fecal contamination. Various methods such as culture-dependent methods, culture-independent gene sequence-based methods, and immunological methods are used to determine bacterial contamination in water sources. As a consequence, determining that the water is not suitable for use by bacteriological analysis indicates that the water is contaminated. This chapter highlights the sanitary quality of aquatic environments, indicator organisms for water quality assessment, detection of bacterial pollution in the water source, and bacterial waterborne diseases.

1. Introduction

Water, which is the main constituent of Earth’s hydrosphere and one of the most essential natural resources for life, is an inorganic chemical substance [1, 2]. It is consumed by societies for various purposes such as human activities (drinking, household, and recreational), agriculture (crop irrigation and food processing), and industry processes [2, 3]. Water-based environments contain the different communities of higher organisms and microorganisms that interact with each other and their environment [4]. Aquatic ecosystems have two main types: the “marine and freshwater ecosystem” [4, 5]. The largest aquatic ecosystem is marine water, which covers more than 70% of the Earth’s surface (estuaries, coral reefs, coastal ecosystems, and oceans). The lotic environment, lentic ecosystem, and wetland habitats are subdivided from freshwater, which constitutes less than 1% of the Earth’s aquatic ecosystems and has a lower salt content [5]. The residues of anthropogenic activities (filling and construction of bridges, canals, dams, roads, and deforestation) industrial and agricultural pollutants discharge into aquatic environments [4, 5]. Especially the freshwater bodies are exposed to wastes and leaks such as detergents, heavy metals, plastic or non-plastic origin compounds, microfibers, etc. [6, 7, 8]. As well as these chemicals and toxic residues reaching aquatic ecosystems, microbial water pollution that occurs even in developed countries is considered one of the major threats to human health across the globe [2, 9]. WHO has reported approximately 46,000 infant deaths and 600 million cases of diarrhea and dysentery per year as a result of contaminated water and insufficient sanitation [10]. The surface water bodies in contact with domestic or sewage wastes from the surrounding area are potentially hazardous ecosystems as carriers of pathogenic microorganisms [2, 11]. Microbiologic monitoring of water quality is critical in terms of detecting, identifying, and quantifying pathogens that cause waterborne diseases [1]. Various microbiologic and molecular techniques including surveillance, detection methods, analysis, and decision-making processes are used in quantitative microbial risk assessment of possible pathogen contamination [12].

Here, we focus on the sanitary quality of aquatic environments, indicator organisms for water quality assessment, techniques used in monitoring water quality, surveillance, quantitative microbial risk assessment, and bacterial waterborne diseases.

2. Sanitary quality of aquatic environments

The physical, chemical and biological characteristics of water define its quality. There is a lot of diversity in these water quality features all over the world. For this reason, natural water sources’ quality in use for various purposes should be determined by means of the water quality parameters [8, 13, 14, 15].

Water is classified as surface and groundwater depending on its origin. Agricultural, industrial, and household activities can contaminate both types of water, exposing them to a variety of contaminants such as fertilizers, heavy metals, oils, pesticides, and toxic compounds (Figure 1).

Figure 1.

Various factors affecting of water quality and disease-causing some bacteria [2].

Potable water, palatable water, polluted water, and infected water are the four types of water quality.

The following are scientific meanings of these types of water quality:

1. Potable water is water that is safe to drink, tastes well, and can be used in the home.

2. Palatable water has a pleasant esthetic and takes into account the existence of chemicals that are not harmful to the health of humans.

3. Polluted water is inappropriate for drinking or domestic use because it contains undesired physical, chemical, biological, or radioactive components.

4. Infected water is that water containing pathogenic organisms [8].

Physical parameters of water quality include turbidity, temperature, color, taste and odor, solids, and electrical conductivity (EC). The chemical water parameters contain pH, acidity, alkalinity, chloride, chlorine residual, sulfate, nitrogen, fluoride, iron and manganese, copper and zinc, hardness, dissolved oxygen, biochemical oxygen demand (BOD), chemical oxygen demand (COD), toxic inorganic and organic substances, radioactive substances. Water quality is also determined by biological parameters such as bacteria, algae, viruses, and protozoa [2, 8, 13, 14, 15]. Considering these parameters, this chapter especially aims to emphasize the bacteriological pollution of water quality.

The presence or absence of live microorganisms can be one of the most beneficial indicators of water quality. Biologists may research organisms in natural waters and measure water quality using an SDI (species diversity index); therefore, a water body with a great number of well-balanced species is considered a healthy environment. The presence of some organisms in aquatic environments is considered an indicator of water pollution [8].

The human intestinal system carries specific population of microorganisms, with coliform bacteria accounting for a large share of this population. Although wastewater contains millions of bacteria per milliliter, the majority of them are safe. The existence of pathogenic bacteria in wastewater can be hazardous when it contains waste from people affected by diseases [8].

3. Indicator organisms for water quality assessment

Nonpathogenic microorganisms which have minimal or no growth in water and can be dependably detected at low concentrations are preferable. Several indicator bacterial species can be utilized in water quality assessment, as explained in the following [3].

Bacteria have been used as indicators of water sanitary quality since 1880 when Von Fritsch characterized Klebsiella pneumoniae and Klebsiella rhinoscleromatis as microbes commonly found in human feces. Percy and Grace Frankland began the first routine bacteriological analysis of water in London in 1885, counting bacteria using Robert Koch’s solid gelatin medium. Escherich also identified Bacillus coli from the feces of breast-fed newborns in 1885. The Franklands proposed in 1891 that the organisms found in sewage must be identified in order to obtain proof of possibly hazardous contamination. Sanitary bacteriologists were employing the “Wurtz method” of counting B. coli via direct plating water samples on litmus lactose agar by using the principle of acid from lactose as a distinguishing characteristic about 1893. With the invention of the Durham tube, the production of gas proved. In 1901, the term ‘coliform’ bacterium was used to describe bacteria that resembled B. coli. Nevertheless, the colony count for bacteria in water was not officially adopted till Report 71. Through the beginning of the 20th century, bacteriologists accepted the sanitary importance of identifying diverse coliforms, as well as streptococci and C. perfringens. MacConkey’s broth which was used to identify lactose-fermenting bacteria that were bile salt tolerant was defined by MacConkey in 1905 [16, 17].

“Standard Methods for the Examination of Water and Wastewater” was first published in 1905. The most appropriate indicator organism for raw drinking water was Escherichia coli. Nevertheless, early-century E. coli detection techniques were not amenable to routine surveillance that allowed for rapid detection of fecal contamination. Consequently, a wider range of organisms related to and containing E. coli, dubbed “coliforms” by Blachstein in 1893, were examined as proxy indicators. Since then, the coliform group has been revised depending on the ability to categorize members based on their genetic structure. Bacterium coli has been renamed E. coli, Bacterium aerogenes has been renamed Enterobacter aerogenes, and the ‘intermediate’ varieties have been classed [17].

The discovery that certain ‘fecal coliforms’ were not of fecal origin, and the development of advanced E. coli testing procedures, have led to the use of E. coli as the ‘favored’ indicator for the identification of fecal contamination [17].

The definition of fecal coliforms has been updated to better reflect the genetic structure of its members and to incorporate newly discovered environmental organisms. After all, fecal coliforms are becoming more commonly known as “thermotolerant” coliforms. This, together with enhanced E. coli detection techniques, has prompted a tendency to use E. coli as a more trustworthy indicator of fecal pollution in drinking water than thermotolerant coliforms.

Escherichia coli, fecal coliforms, and/or Coliforms are the indicator bacterias currently used for drinking water monitoring in developed nations, though the belief of indicator organisms as the primary source of data about the safeness of drinking water is being questioned in so many states. The World Health Organization (WHO) proposed E. coli as the primary indicator of fecal contamination in 2003 [17, 18].

Coliforms are all facultative anaerobic, non-spore-forming, gram-negative, rod-shaped bacteria, oxidase-negative, fermenting lactose to acid and gas at 35°C in 48 h or members of the Enterobacteriaceae that are β-galactosidase positive [16, 17]. The coliforms contain innocuous E. coli and Enterobacter, as well as the most prevalent intestinal bacteria and rare pathogens such as Klebsiella, Citrobacter, Kluyvera, and Leclercia genera, and also some members of the Serratia genus. These microorganisms are utilized as a fecal pollution indicator in water since they are found in the intestinal tracts of homeothermic animals and seem to have sanitary importance [2, 8, 16, 17, 18, 19].

Fecal coliforms are bacteria that can grow and ferment lactose with the formation of acid and gas in EC broth (Escherichia coli Broth) at 44.5°C within 24 h. This group associated with fecal pollution in warm-blooded animals is known “thermotolerant coliforms”. It also includes E. coli, is predominantly fecal, as well as other species, which are called non-E.coli such as Klebsiella, Enterobacter, and Citrobacter. Septic systems and sewer, run-off from dairy, feedlot, and farming sites, stormwater, and straightly defecating of livestock to water can introduce the fecal coliforms enter to rivers and streams [3, 8, 13, 16, 17, 18, 19, 20].

Fecal streptococci are Gram-positive coccoid bacteria, which were being studied as a key of indicator microorganism contamination. However, difficulties in distinguishing fecal from non-fecal streptococci hampered their utilization. Suckling had offered 4 main pieces of evidence in support of fecal streptococci in 1943: [16].

1. Feces of humans and other warm-blooded animals in quite great quantities.

2. Existence in contaminated waters and wastewaters.

3. No presence of environments, pure waters, and pristine soils free of animal and human life.

4. Permanence without reproduction within the environment.

Enumeration of Fecal Streptococci occurred widespread after the useable of Slanetz and Bartley’s selective medium in 1957. Thenceforth, many mediums have been suggested to enhance specificity for fecal streptococci and enterococci. Fecal streptococci are characterized by Streptococcus equinus, Streptococcus bovis, and divers Enterococcus spp. The enterococci are the primary indicators of fecal contamination among the fecal streptococci. Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, and Enterococcus durans, are the most common intestinal enterococci. Additionally, certain Streptococcus species including Streptococcus bovis and Streptococcus equinus, and other Enterococcus species may be observed on occasion. On the other hand, these streptococci do not live long in water. Because of this enterococci can be used to detect fecal pollution in water [3, 16, 17, 18, 19].

Escherichia coli is a thermotolerant coliform that produces indole from tryptophan, however, it is currently also described as coliform that includes the glucuronidase. Furthermore, E. coli is identified enzymatically by the absence of urease [17, 18, 19].

A wide range of species has existed as fecal contamination indicators. Unfortunately, none have proven to be completely efficient in this function yet. Many, such as Enterococci, have a significant environmental occurrence, whilst others, such as Clostridium perfringens, are anaerobic through nature or need difficult isolation procedures, such as bacteriophages [16, 17, 19].

4. Detection of bacterial pollution in the water source

Several recent studies have been reviewed culture-dependent methods (heterotrophic plate count, most probable number method, membrane filtration method, and defined substrate/enzyme methods), culture-independent gene sequence-based methods (microbial source tracking, polymerase chain reaction, fluorescence in situ hybridization, and next-generation sequencing, etc.) and immunological methods used to assess bacterial pollution and microbial load in water bodies [17, 21].

4.1 Culture-dependent methods (colorimetric, counting, and fluorimetric analyses)

The most common approaches for the determination of bacterial pollution in environment samples are based on bacterial culturing methods that usually performed biochemical methods required time approximately three days or even longer [22]. The culture-dependent methods applied for the detection of indicator bacteria are schematically shown in Figure 2.

Figure 2.

Some examples of culture-dependent detection methods of indicator bacteria [23, 24, 25, 26].

The heterotrophic plate count tests (standard plate count/agar plate count) are widely used for estimating the number of live heterotrophic bacteria in various water bodies is a simple procedure that shows the earliest sign of pollution. HPC test methods (pour plate and spread plate methods) are based on incubating at temperature conditions ranging from around 20 to 40°C of agar plates absorbing a small volume of sample or diluted sample (0.1 to 0.5 mL) All colonies grown on the agar surface and arose from pairs, chains, clusters, or single cells are enumerated and expressed as colony-forming units (CFU) [21, 27].

The multiple tube fermentation procedure is firstly adopted as a bacteriological standard by USA Public Health Service Drinking Water Standard in 1914. This method is indicated the presence of total coliforms, indicators of organic pollution in water sources, and is now referred to as the Most Probable Number (MPN). MPN is illustrated by Prescott et al. [28]. consists of inoculation steps into 15 tubes consisting of 5 tubes for each of three dilution factors (0.1, 1, and 10 mL) each tube containing an inverted Durham tube. This is for detecting total coliforms in the family Enterobacteriaceae relying on the production of acid and gas during lactose fermentation varying with the composition of the media [16, 17]. The coliform pollution in samples is stated as the most probable number (MPN) provided a statistical estimation according to the MPN table [22].

Fluorimetric or colorimetric assays based on specific enzymatic activities are rapid analytical techniques applied for the assessment of total and fecal coliform in water. The enzymatic detection method utilizes the hydrolyzable chromogenic and fluorogenic substrates in the recognition of 𝛽-galactosidase in total coliform or 𝛽-glucuronidase in E. coli [2]. Ortho-nitrophenyl-𝛽-D-galactopyranoside (ONPG) or chlorophenol red-𝛽-D-galactopyranoside (CPRG) are substrates that show a distinct color change in the medium following the 𝛽-galactosidase enzyme hydrolysis. A fluorogenic substrate, 4-methylumbelliferyl-𝛽-D-glucuronide (MUG), is hydrolyzed by specific bacterial enzymatic activity to a fluorescence product viewed under ultraviolet (UV) light [29].

The membrane filters in conjunction with Endo-broth for enumerating total coliforms were initiated and used by Mueller in Germany, in 1943 [16]. The water sample is filtered by a membrane with a pore size of 0.45 mm and density may be calculated by counting bacterial cells on the membrane incubated on agar plates. The sensitivity of filtration methods is significantly impressed by the type and quality of membrane filter and the number of colonies.

These culture-based methods that required excessive time (18–96 h) and intensive training for confirmation and verification steps in the detection and quantification of water-origin bacterial pathogens are low sensitivity. Furthermore, the existence of viable but non-culturable (VBNC) bacteria and bacterial pathogens groups at low concentrations in a large volume of water bodies limit efficient quantification and cause false-negative results [3, 12, 30]. Therefore, culture-independent methods having important requirements such as specificity, sensitivity, reproducibility of results, speed, automation, and low cost for reliable analysis are increasingly developing in the last few years [31].

4.2 Immunological methods

Immunological methods are antibody–antigen interactions based on the specific binding affinities of antibodies to specific antigens These methods used polyclonal and monoclonal antibodies include different methods such as lateral flow tests (immunochromatographic assays), SPR (Surface Plasmon Resonance), ELISA (Enzyme-Linked ImmunoSorbent Assay), immunofluorescence, chips, and Western blots etc. (Figure 3). The specificity and sensitivity of each technique having the rapid application and specific device requirement depend upon the antibody. However, limitations such as reducing the cell surface antigens, cross-reactivity, false-negative results, and no indication of the viability of organisms for these methods used in the detection of aquatic bacterial pollution are present [12, 21].

Figure 3.

A view of immunological approaches for bacterial quality of water sources [32, 33, 34, 35, 36].

4.3 Culture-independent genetic methods

Microbial source tracking (MST) has performed at the end of the 20th century and is a molecular technique to detect the dominant sources of fecal contamination in environmental water samples. Microbial source tracking incorporates unique genetic sequences to specific fecal species from fecal sources (e.g. human, dog, cattle) contacted with water bodies The basic approach of MST is the identification of the fecal source using signature molecules (markers) of particular host-associated microorganisms [3, 37]. This method is tested by two basic strategies: 1- library-dependent analyses based on isolation and typing of fecal indicator bacteria for some identifying properties such as their phenotypic (antibiotic resistance, carbon source utilization, etc.) or genotypic fingerprints, and 2- library-independent analyses that required target genes of specific bacterial species such as variable region of the 16S rRNA [37].

Polymerase chain reaction (PCR) has been applied for the detection and identification of total coliform in 100 ml water samples by a screening of the lacZ (developed by Bej et al., wecG, and 16S rRNA genes in the field of water monitoring [2, 38]. Horakova et al. analyzed 𝛽-d -glucuronidase (uidA), lactose permease (lacY), 𝛽-d -galactosidase (lacZ), and cytochrome bd complex (cyd) four target DNA sequences for specific detection of E. coli cells in the water samples. Multiplex PCR, Nested PCR, In situ PCR, and Quantitative real-time PCR (qPCR) methods following the general principle of PCR are used to determine fecal indicator bacteria and microbial load in waters [17, 39]. Detection limits of PCR methods for different water sources are presented in Table 1.

PCR detection method	Water-borne bacterial pathogen	Detection limit	Water source
Conventional PCR	E. coli; Enterotoxigenic E. coli (ETEC)	1 cfu/100 mL; 4 cfu/mL	Contaminated tap water,; Water samples spiked by ETEC; and nonpathogenic E. coli.
Multiplex PCR	EHEC, Shigella sp., Vibrio parahaemolyticus,; P. aeruginosa and Salmonella sp.	10^1 cfu, 10^2 cfu, 10^2 cfu, 10^2 cfu and 101 cfu/mL	Polluted water and natural water
Quantitative PCR (qPCR)	L. monocytogenes,; V. cholerae,; V. parahaemolyticus,; Pseudogulbenkiana; sp., S. typhimurium,; S. flexneri,; C. perfringens and pathogenic E. coli	From 10^2 to 10^4 cells per ca. 200 mg fecal samples of pathogens 100 cells/L	Spiked environmental water samples (pond) and a natural freshwater lake.
Real-time PCR	V. cholerae	1 cfu/100 mL	Ballast water.

Table 1.

Limitations of PCR methods for different water sources [12].

Fluorescence in situ hybridization (FISH) reports the presence of fecal contaminant and viable indicator bacteria but nonculturable (VBNC) through hybridization of the sample with rRNA oligonucleotide probes labeled with a fluorescent dye. FISH allows an enumeration of microbial cells in seawater, freshwater, and wastewater samples to obtain qualitative and quantitative results by using fluorescence microscopy, flow cytometry, or confocal microscopy [40].

The most novel approach in the microbial investigation of water quality is Next-generation sequencing (NGS) based on the amplification of environmental DNA samples. This method sequenced hypervariable regions (V1, V2, and V4) of unique small subunit SSU rRNA in the microbial communities provides high quality to evaluate fecal pollution [3].

A Diagram of molecular approaches based on genetics is represented in Figure 4.

Figure 4.

Molecular strategies for detecting of water quality [41, 42, 43, 44].

5. Bacterial waterborne diseases

The discharge of waste and stormwater to the surface, coastal, and groundwater, as well as agricultural flows including animal and human waste and nutrients, is raised the microbial pollution of aquatic habitats [30, 45]. Some bacteria among those can ubiquitously occur in many water systems and commonly are recognized water-borne pathogens. Bacterial pathogens including several groups of enteric and aquatic bacteria are classical etiological agents of water-transmitted diseases globally [2, 46]. These pathogens associated with certain infections in humans and animals have been responsible for worldwide tremendous morbidity and mortality [2, 19, 47]. A number of reasons such as the excessive increase in population, globalization of travel and commerce as well as contamination of drinking water are caused the appearance again and again of water-associated diseases. Especially, the lack of technological and financial resources in developing countries contributes to emerging waterborne outbreaks [12]. Children under 5 years of age are particularly influenced by diarrheal infections transmitted by contaminated drinking water in African and Asian countries. It is also referred to has no access to safe and clean drinking water and dying more than 1.5 million children per year from contaminated drinking water [19].

Vibrio cholerae and Salmonella enterica serovar Typhi (now known as Salmonella typhi) were identified as the first water-borne pathogens in the 19 th century [2]. A list of declared bacterial water-transmitted diseases from the 19th century to the present is shown in Table 2.

Bacterial agents	Associated diseases	References
Campylobacter spp., Campylobacter jejuni	Diarrhea, Gastroenteritis	[12, 19]
Escherichia coli, particularly enterohemorrhagic E. coli (EHEC), and others such as enteropathogenic (EPEC), enterotoxigenic (ETEC), and enteroinvasive (EIEC)	Acute diarrhea, bloody diarrhea, and gastroenteritis	[12, 19]
E. coli O157:H7	Bloody diarrhea, hemolytic uremic syndrome	[5]
Legionella pneumophila and related bacteria	Acute respiratory illness, pneumonia (legionellosis)	[12, 19]
Salmonella enterica serotype Typhi	Typhoid fever, paratyphoid, salmonellosis	[19]
Shigella spp.	Bacillary dysentery	[19]
Vibrio cholerae	Cholera, Gastroenteritis	[12, 19]
Yersinia enterocolitica	Gastroenteritis/Yersiniosis	[1, 19]
Helicobacter pylori	Abdominal pain, peptic ulcers, gastric cancer, and chronic gastritis,	[5, 12]
Burkholderia pseudomallei	Meliodosis	[12]
Non-tuberculous mycobacteria	Pulmonary disease, skin infection	[12]
Pseudomonas aeruginosa	Infections on the lungs, urinary tract, and kidney, inflammation, and sepsis	[12]

Table 2.

Some potential examples of the bacterial water-transmitted diseases.

6. Conclusions

The aquatic environments are important ecosystems threatened by harmful pollutants (metals, agrochemicals, nanoparticles, radioactive elements, volatile organic compounds, personal care products, household products, industrial solvents, and waterborne pathogens) due to anthropogenic residues and lack of sanitation. For this reason, the regular control, monitoring, understanding, and evaluation of water quality is a global concern for environmental and public health safety. One of the most parameters of water quality in terms of health risk assessment is, indicator bacteria are key to determining microbiological perspective. Culture-dependent methods, molecular methods, and immunological techniques for estimating bacterial load and populations in water systems, culture-dependent are routinely utilized. Despite having several disadvantages (costly, excessive time consuming, the lack of protocol and sample processing standardization, etc.) of each method, molecular techniques are the most effective strategies to identify causative agents to water-related diseases and waterborne outbreaks and the distribution of proxy bacteria in water bodies.