Relationships between FIB and pathogens - Faecal indicator bacteria

2.3 Faecal indicator bacteria

2.3.1 Relationships between FIB and pathogens

FIB are common in the intestine of all warm-blooded animals (healthy, asympto-matic carriers of some pathogens and infected hosts), but pathogens are common only to the infected host (symptomatic, asymptomatic and recently recovered).

Further, the host ranges of FIB are wide, FIB are released from all warm-blooded animals, including human beings, but certain pathogens, like human-infecting vi-ruses, are released mainly from infected humans. Therefore, FIB are detected in higher numbers in water bodies than pathogens. A good correlation between enter-ic viruses and FIB has been reported at bathing sites with human faecal contamina-tion and a good correlacontamina-tion between zoonotic pathogens and FIB can be at bathing sites with animal faecal contamination (Harwood et al. 2014). The illnesses from autochthonous microbes, such as Vibrio spp. and toxins produced by Cyanobacteria, may have poor relationships with FIB counts (Hlavsa et al. 2014, Islam et al. 2020).

Various other reasons for poor relationships can be found, such as different decay rates and transport characteristics, that may affect the relationship between patho-gens and FIB (Wu et al. 2011).

The persistence or probable growth of FIB in environmental habitats, such as soil, sediments, and vegetation, has been reported (Whitman et al. 2003, Badgley et al. 2011, Byappanahalli et al. 2012). Such environmental habitats may work as a source and sink of FIB (Ishii and Sadowsky 2008, Byappanahalli and Ishii 2011, Byappanahalli et al. 2012). The release of FIB from the environmental source has poor relation with enteric pathogens and poses a false positive alarm concerning the human health risk (Badgley et al. 2011). Environmental disturbances due to tides, winds, animal movements, and anthropogenic disturbance, such as bathing and water sports, can stir FIB from the sediment and aquatic vegetation and may increase FIB counts in water (Boehm et al. 2009). In contrast, the long persistence of enteric pathogens over FIB in an environmental habitat also weakens the relation between FIB and pathogens. Due to a false-negative FIB alarm, pathogens may jeopardize the health of beach users (Anderson et al. 2005, Badgley et al. 2010). Fur-ther, currently used FIB cannot differentiate the source of contamination. Therefore, the criteria for ideal FIB cannot yet be fulfilled completely.

The pathogens, mostly bacteria, viruses, and protozoa, have a wide variation in the taxonomic range, cell structure, morphology, and physiology and they can have different decay rates than FIB (Anderson et al. 2005, Byappanahalli et al. 2012, Lutz et al. 2013). Therefore, these wide taxonomic ranges of microbes might have a dif-ferent response towards environmental stresses, such as unfavourable pH values, solar radiation, salinity, predation and temperature and nutrients concentration (Anderson et al. 2005, Korajkic et al. 2018). FIB can be more sensitive to inactivation in the wastewater treatment process and by sunlight, than viruses and protozoan parasites (Sinclair et al. 2009). However, in fresh faecal contamination, there can be a high correlation between FIB with a human enteric virus and parasitic protozoa (Hartel 2011).

25 2.3.2 Coliform bacteria and E. coli

Coliforms are a group of bacteria that express the enzyme β-D-galactosidase (ISO 9308-2 2012). In the ISO 9308-2 method, coliform positive wells produce a yellow colour and are recognized by visual inspection. These are a Gram-negative, non-spore-forming, oxidase negative, rod-shaped bacteria that can grow in aerobic or facultatively anaerobic conditions in the presence of bile salt (Chao 2006, ISO 9308-2 2012). The earlier definition, based on lactose fermentation, defines coliform as a group of bacteria having the capacity to ferment lactose into gas and acid within 48 hours at 32-35°C (APHA 1989, Chao 2006). These bacteria belong to different genera within the Enterobacteriaceae family, namely: Escherichia, Klebsiella, Citrobacter, Haf-nia, and Enterobacter. This group of coliform bacteria was used for a long time as FIB for the regulatory monitoring of bathing water quality (EC 1976). However, the use of total coliforms as FIB has some limitations, such as their ability to grow in a natu-ral environment (Carrillo et al. 1985, Byappanahalli et al. 2006) and a lack of correla-tion with faecal pathogens and numbers of bathing water illness cases (Wade et al.

2003, Korajkic et al. 2018). The current bathing water monitoring method (EC 2006) does not include this group of bacteria for regulatory monitoring.

Faecal coliforms are a subgroup of total coliforms that are more specific to the faecal origin. The members of this group of bacteria are capable of growing in the presence of bile salts. These are oxidase negative, and produce acid and gas from lactose within 48 hours at 44 ± 0.5°C (APHA 1989). Faecal coliform bacteria can tolerate higher temperatures than other members of coliforms, so faecal coliforms are also called thermotolerant coliform bacteria. Some members of faecal coliform bacteria, like Klebsiella, can persist for a long time in environmental water.

E. coli is a major species of faecal coliform group, which is the most reliable among the coliform group as FIB (Korajkic et al. 2018). E. coli expresses both galactosidase and glucuronidase enzymes (ISO 9308-2 2012). The β-D-glucuronidase activity can be measured with fluorescence produced under ultra-violet light (365 nm; ISO 9308-2:2012). Further, E. coli is a species among the group of coliform bacteria which can produce indole from tryptophan within (21±3) h at (44±0.5) °C. It provides a positive result in the methyl red test and can decarbox-ylate 1-glutamic acid, but is not able to produce acetyl methyl carbinol, utilize cit-rate as the sole source of carbon or grow in KCN broth. However, some strains of E.

coli, such as E. coli O157:H7 express only β-D-galactosidase activity, but do not have β-D-glucuronidase activity. E. coli ferments lactose at 44 °C and produces indole from tryptophane (Chao 2006, ISO 9308-2 2012). It is a primary indicator of choice of inland bathing water quality monitoring (Wade et al. 2003) and is also applied to coastal bathing water monitoring. E. coli has been used as a FIB for regulating the monitoring of bathing and recreational water quality (EC 2006, USEPA 2012).

2.3.3 Enterococci and intestinal enterococci

Enterococci (ENT) are Gram-positive, non-spore-forming, obligatory fermentative, chemoorganotrophic coccoidal bacteria, belonging to the genus Enterococcus (Boehm and Sassoubre 2014). This group of bacteria grows at a temperature range of 10°C - 45°C in 6.5% NaCl at pH 9.6 and survives for 30 minutes at 60°C, hydro-lyzes aesculin, is resistant to sodium azide and reduces triphenyl tetrazolium chlo-ride (Byappanahalli et al. 2012). ENT are a primary indicator of choice for the toring of coastal bathing water (Wade et al. 2003) and are also applied to the toring of inland bathing water. ENT have been used as FIB for regulating the moni-toring of bathing and recreational water quality (EC 2006, USEPA 2012).

The term enterococci and intestinal enterococci (iENT) have been used inter-changeably (Leclerc et al 1996, Byappanahalli et al. 2012). However, iENT are a sub-group of ENT majorly belonging to four species Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus hirae (WHO 2003). These species grow at 44 °C at aerobic conditions, hydrolyze 4-methylumbelliferyl-b-D-glucoside in the presence of thallium acetate, nalidixic acid and 2,3,5-triphenyl-tetrazolium chloride (ISO 7899-2 2000). Among them, E. faecium and E. faecalis have a high prevalence in human faeces (Moore et al. 2006; Layton et al. 2010). Enterococcus is a large genus, and not all species originate from faeces (Byappanahalli et al. 2012). For example, Enterococcus mundtii, Enterococcus casseliflavus, Enterococcus aquimarinus, and Entero-coccus sulfureus are reported from vegetation sources (Mundt and Hinkle 1976, Moore et al. 2006, Byappanahalli et al. 2012).

2.4 ALTERNATE FIB

Bacteria, protozoa, and viruses of a wide taxonomic variation have a different cell structure, size, and defence mechanism against external stress factors (Field and Samadpour 2007, Griffith et al. 2016), so their decay rates in various environmental stress conditions are different (Griffith et al. 2016, Korajkic et al. 2018). So, FIB used for monitoring the microbial quality of surface water does not always relate to pathogens and parasites (Savichtcheva and Okabe 2006). That is why, different alternate indicators, such as F+ RNA coliphages, somatic coliphages, and bacterial genera Bacteroides, Prevotella, Catellicoccus, Clostridium, Bifidobacterium, Staphylococcus and Brevibacterium, have been used for the monitoring of water quality (Savichtche-va and Okabe 2006, USEPA 2012, Fujioka et al. 2015, Korajkic et al. 2018).

Viruses generally have longer survival rates in ambient environments than FIB (Thurston-Enriquez et al. 2003). Viruses can be detected in surface water, even when the FIB numbers are below the safe limit according to current monitoring protocols (Sinclair et al. 2009, Kauppinen et al. 2017). Further, a stronger correlation between F-specific coliphages and somatic coliphages with GI illness than bacterial indica-tors has been demonstrated (Wade et al. 2003, Griffith et al. 2016). Such findings

27 justify the need for a separate viral indicator for the regulatory monitoring of bath-ing water (USEPA 2015, WHO 2018). Currently, F-specific and somatic coliphages have been used as an alternate feacal indicator (Griffith et al. 2016). Coliphages have similar physical structure, composition, morphology and survival characteristics in the environment; and these are suggested as a potential viral indicator for faecal contamination for monitoring bathing water (USEPA 2015). F-specific coliphage is one of the most used virus indicators for water quality testing (USEPA 2015). Its detection and quantification methods are simple, reliable, rapid, and inexpensive (USEPA 2015). Somatic coliphages are more persistent against environmental stress factors and have a higher concentration in faeces than F-specific coliphages (USEPA 2015). Norovirus resembles more F-specific coliphages than somatic coliphages due to having single-strand RNA, while adenovirus resembles more somatic than F-specific coliphages due to having double-stranded DNA (USEPA 2015).

However, the current FIB cannot differentiate the source of faecal contamination (Stoeckel and Harwood 2007). Strict anaerobic gut bacteria genera, such as Bac-teroides, Catellicoccus, Brevibacterium and Prevotella that are highly host-specific, are used as alternative faecal indicators and for microbial source tracking (McLellan and Eren 2014). Also the bacterial species Clostridium perfringens is highly resistant to environmental stress factors and have a limited potential to proliferate in the ambient environment. This species has been used as an alternate faecal indicator for the monitoring of bathing water (Fujioka and Shizumura 1985, Okabe and Shimazu 2007, Fujioka et al. 2015).

During bathing seasons, a high number of bathers may visit bathing sites in a single day. There can be a high chance of a cross-contamination of pathogens among bathers. However, the currently used FIB cannot indicate such cross con-taminations. The bacterial species Staphylococcus aureus has been studied as an indi-cator of direct contamination from bathers to bathers (Elmir et al. 2007). The species has a high survival rate in coastal water. It is more resistant to chlorine and salinity than bacteria from the total coliform group (Elmir et al. 2007). Further, a positive correlation between the numbers of S. aureus in water with skin, ear, and respirato-ry tract illness was demonstrated at a coastal bathing site (Elmir et al. 2007). In addi-tion to all the above-menaddi-tioned microbial groups as an alternate indicator, direct enumeration of pathogens, mainly Salmonella spp. and enteric viruses, are also con-sidered as a sign of faecal contamination (Savichtcheva and Okabe 2006).

3 METHODS FOR MICROBIOLOGICAL

BATHING WATER QUALITY CHARACTERIZA-TION

The microbial quality of bathing water has been monitored for decades by measur-ing indicator bacteria that are common in the gut of warm-blooded animals, includ-ing human beinclud-ings. Historically, microbial monitorinclud-ing was started with microscopic techniques, but now culture-based methods and molecular methods are the two most used water quality monitoring methods. The strengths and weaknesses of variants of these two most commonly used methods are listed in Table 3.

Table 3. Strengths and weaknesses of different microbiological monitoring methods. MF= membrane filtration, MPN = most probable number, PCR = polymerase chain reaction, qPCR= quantitative poly-merase chain reaction, RT-qPCR= reverse transcription-quantitative polypoly-merase chain reaction.

Method Strengths Weaknesses

Culture MF meth-ods

Widely accepted gold standard, econom-ically cheap, highly standardized, and easy to operate and interpret

Long incubation time, not suitable for unculti-vable microbes, not suitable for samples hav-ing high suspended particles

Culture MPN methods

Widely accepted gold standard, econom-ically cheap, highly standardized, and easy to operate and interpret

Long incubation time, not suitable for unculti-vable microbes, sometimes identification can be subjective due to the phenotypic method

PCR Highly specific Not quantitative

qPCR (DNA target)

Fast, highly specific, can enumerate different microbes by changing target primers.

May detect dead and non-viable microbes, high installation and operation costs, even a small laboratory mistake can alter the result RT-qPCR

(RNA target)

Count only viable cells, no need to incu-bate, highly sensitive, can differentiate the source of faecal contamination

Cell quantification is not possible

High-throughput sequenc-ing

Provides information on a large group of microbes at the same time, gives an idea about the microbial community structure

Resource intensive. Not fully developed yet.

The taxonomic genes of some species are highly conserved, so cannot identify the genus and species level

3.1 CULTURE-BASED METHODS

Culture-based methods are the accepted gold standard for enumerating FIB and many pathogens. These methods are relatively inexpensive, highly standardized, and easy to operate and interpret. The culture-based methods can be different types, but membrane filtration based, the most probable number (MPN) method and chromogenic substrate methods are most widely used. The targeted indicators are selectively isolated and incubated in nutrient-rich media (Niemelä et al. 2003, Pitkänen et al. 2007). However, these methods are often criticized for enumerating

29 only the viable organism. Even within the viable group, not all of them are cultura-ble (Pitkänen et al. 2013). Further, these methods require a relatively long incuba-tion time, ~18-48 hours at the minimum, depending on the target FIB. These meth-ods rely on biochemical or immunological methmeth-ods of identification. Sometimes, such identification can be subjective and have personal biases (Niemelä et al. 2003, Pitkänen et al. 2007).

Membrane filtration (MF) is a widely used water sample concentration tech-nique utilized before the culture-based quantitation of microbial targets (ISO 7899-2 2000). By using this method, indicator bacteria from water samples are initially concentrated into a membrane filter with a help of filtration, and then the mem-brane is transferred onto a petri dish having a solid selective growth medium spe-cific for the target organism (ISO 7899-2 2000). Then the petri dish containing the culture medium with the membrane filter is incubated for about 35-45 ⁰C, depend-ing on the medium used. The number of colonies grown on the membrane filter is counted and expressed as the colony-forming unit CFU/100 ml sample (ISO 7899-2 2000). The multiple tube fermentation (MPN) method is the next common culture-based method used for microbial enumeration. As the MF method is not suitable for the water samples having high turbidity, the MPN method overcomes the limi-tation. In this method, water samples are poured into a liquid medium, and the microbial counts are expressed in MPN/100ml (ISO 9308-2 2012). Viruses and bacte-riophages are concentrated from water samples by using electropositive filters, electronegative filters, and ultrafilters (Cashdollar and Wymer 2013). Host cells are needed for culturing viruses and the viruses can be isolated from the resulting plaques (Cashdollar and Wymer 2013).

3.1.1 E. coli monitoring

The membrane filtration-based ISO 9308-1 2000 method and the miniaturised most probable number (MMPN) based ISO 9308-3 1998 method are official methods for enumerating E. coli; according to bathing water directive (BWD EC 2006). However, the ISO 9308-1 (2000) method has been modified completely to ISO 9308-1 (2014).

The earlier version of the method operated on two steps and used TTC Tergitol® 7 agar and rapid test using TSA/TBA agar. The recent version of ISO 9308-1 (2014) of the method uses Chromogenic Coliform Agar (CCA) media. The ISO 9308-1 (2000) method has been criticized as not being suitable for monitoring environmental wa-ter samples having a high background flora (Jozić et al. 2018, WHO 2018). The re-cent version is intended mainly for monitoring the high quality of drinking water (Jozić et al. 2018, WHO 2018). The next official method for enumerating E. coli, ISO 9308-3 1998 is not used in laboratories serving local health protection authorities of Finland. Both methods need about 48-72 hours to confirm the results.

Colilert-18 Quanti-Tray (ISO 9308-2 2012) is relatively more rapid than the refer-ence methods and gives a result within 18 hours. It is an accepted method for use in monitoring the quality of drinking water in many countries (Niemelä et al. 2003, Pitkänen et al. 2007). In both Colilert-18 and MMPN methods, the detection of E. coli

is based on the fluorogenic reaction (positive for β-glucuronidase) (Lebaron et al.

2005, Valente et al. 2010). The Colilert-18 method also detects coliform bacteria, based on chromogenic reaction, and the E. coli result is recorded by both chromo-genic and a fluorochromo-genic reaction to occur and are detected in the same well (Nie-melä et al. 2003, Lebaron et al. 2005, Valente et al. 2010). The MMPN method enu-merates E. coli, but no other coliform bacteria groups (Lebaron et al. 2005, Valente et al. 2010).

3.1.2 Enterococci monitoring

Different varieties of techniques are available for ENT enumeration (Domig et al.

2003). Most of the available methods are based on Slanetz and Bartley (S&B) agar (Slanetz and Bartley 1957) and the Kanamycin Aesculin Azide (KAA) agar (Domig et al. 2003). EUBWD has assigned two methods; ISO 7899-1: 1998 and ISO 7899-2:

2000 for the selective isolation and enumeration of ENT from bathing water. The first one (ISO 7899-1 1998) is based on the MPN method, using the miniaturized 96-well system to enhance precision. The Miniaturized MPN enumerates iENT on a basis to their capacity to grow at 44 ± 0.5 °C and of hydrolyzing 4-methylumbelliferyl-b-D-glucoside in the presence of thallium acetate, nalidixic acid, and 2,3,5-triphenyltetrazolium chloride, in the liquid medium. The presence of iENT is visualized by the emission of fluorescence in 36-72 h. The second method (ISO 7899-2 2000) is based on membrane filtration and confirms iENT in two steps.

At first, the bacteria retained on the membrane filter are incubated for 44 ± 4 h at 36

±2 °C on a S&B medium. The triphenyltrazolium chloride (TTC) in S&B medium is reduced to formazan and forms red colonies. All new red or maroon coloured colo-nies are accepted as presumptive ENT. The presence of iENT is then confirmed on bile esculin azide (BEA) agar (incubating for 2 h at 44 ± 0.5 °C). ENT hydrolyze es-culin to esculetin, react with ferric citrate in the medium to produce a black phenol-ic iron-complex giving esculinase-positive colonies a brown-black halo. The iENT is confirmed, based on dark brown to black colonies produced on a BEA agar medi-um.

3.2 MOLECULAR METHODS

Methods, such as polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), next-generation sequencing (NSG), nested PCR, digital PCR (dPCR), multi-plex PCR, and microarrays, are popular molecular tools in health-related water microbiology (Vierheilig et al. 2015, Zhang and Liu 2019). The use of molecular methods has made it possible to enumerate the large range of pathogens directly from surface waters without culturing them. Monitoring all microbes with the cul-ture-based method is a great challenge as most microbes in the natural environ-ments are difficult to culture.

31 Molecular methods, mostly qPCR and RT-qPCR, ensure more rapid results than culture-based methods, due to a lack of a microbial multiplication phase.

Rapid methods have high importance for the monitoring of bathing water quality, as the microbial quality of bathing sites may change drastically within 24-48 hours, i.e. during incubating and enumerating FIB with the culture-based methods. In the United States, U.S. EPA has approved a qPCR-based enterococci gene copy enu-meration method for the monitoring of regulatory bathing water (USEPA 2012).

The Entero1 assay is the recommended qPCR assay for the purpose (USEPA 2012).

However, the Entero1 primers targets all known species of Enterococcus genera (Ludwig and Schleifer 2000, Haugland et al. 2005), which is equivalent to the total enterococci enumerated with membrane-Enterococcus Indoxyl-β-D-Glucoside (mEI) agar method (USEPA 2012). Earlier studies reported that molecular markers can predict bathing water-related human health risks and the presence of a human virus (mainly norovirus) better than the culture-based methods used in the USA (Wade et al. 2008, Schoen et al. 2011). A strong correlation between culturable cells of E. coli with its qPCR markers was recorded earlier (Shrestha and Dorevitch 2019).

In comparison to culture-based methods using phenotypic characterization, the molecular qPCR methods can have high specificity, as they use genotypic character-ization (Savichtcheva and Okabe 2006). However, the partial confirmation may cause biases during reading the positive cases (Niemelä et al. 2003, Pitkänen et al.

2007) in both the phenotypic and genotypic confirmation. Most of the molecular tools (PCR, qPCR, and amplicon-based NGS) use the 16S rRNA gene for the identi-fication of microbes. These genes are highly conserved among bacteria and archaea and are useful for taxonomic identification of microbes. Instead of this gene,

In document Monitoring microbial quality, contaminant sources and bacterial communities of bathing water in Finland (sivua 26-0)