Sustainable Building Engineering 2021/22

Environmental Chemistry and Biology

Environmental Biology

Lecture 2. Microbiology of water

Beata Mądrecka-Witkowska, PhD

Institute of Environmental Engineering and Building Installations
Faculty of Environmental Engineering and Energy
Poznan University of Technology
 • Autochthonous and allochthonous
 microorganisms of surface waters
• Indicators of microbiological contamination
Microbiology of water

of water • Polish and international standards for the

quality of drinking water
• Diseases caused by microorganisms living in
surface waters and in sanitary installations
 Aquatic environments

Water covers about 75% of the Earth

Freshwater environments
0.005-0.06% water salinity Brackish environments*
(6% of surface water) 0.06-3.2% water salinity

Springs Streams Rivers Lakes* Estuaries*

Ponds Wetlands Seas Oceans

Marine environments
3.2-3.8% water salinity

(Libudzisz et al., 2007)

 Factors influenced microbial growth in surface water
Factors

Abiotic factors – factors that are not related to Biotic factors – caused by the presence of other
living organisms organisms.

(Libudzisz et al., 2007)

 Factors influenced microbial growth in surface water
Abiotic factors – factors that are not living organisms
1) Physical factors e.g.
 Light
 Water temperature
 Water turbulence
 Water turbidity
2) Chemical factors e.g.
 Availability of organic and mineral substances which are source of food
 Salinity
 Presence of pollutants and toxic chemical compounds (e.g. heavy metals,
pesticides, herbicides, antibiotics)
 Composition and concentration of dissolved gases (e.g. oxygen concentration)
(Libudzisz et al., 2007)
 Factors affecting microbial growth in surface water
Biotic factors
Commensalism – it is very common
relationship in microbial world, literally means water – upon the sediments
"eating from the same table”.
Methanotrophs
Metabolic products of one microbial population
are used by another microbial population
without either gain or harm for the first
population.
Example: Methanogens
(representants of Archaea) live in anaerobic
conditions in the surface layer of the
sediments, in the bottom of the various
waterbodies. They produce energy for Carbon dioxide reduction
metabolic processes by reducing carbon
dioxide to methane (CO2 to CH4).
CO2 + 4 H2 → CH4 + 2H2O
Methanotrophs (representants of Bacteria)
live in aerobic conditions in the water, upon the
sediments. They oxidize methane back to
surface layer of the sediments
carbon dioxide to produce the energy.
The product of the reaction carried out by one microorganism
is the substrate in the reaction carried out by the other
microorganism.

(Libudzisz et al., 2007)

 Factors influenced microbial growth in surface water

Biotic factors
• Mutualism - is a long-term relationship between individuals of
different species where both individuals benefit.
e.g. Coral reefs are the result of mutualism between polyp’s of
corals and various types of algae which live inside them e.g.
dinoflagellates.
Most of corals obtain the majority of their energy and nutrients
(e.g. glucose, glycerol, and amino acids) from
photosynthetic unicellular algae that live within their tissues.
Algae benefit from a safe place to live and consume the polyp's
carbon dioxide, phosphates and nitrogenous waste.
Symbiont and host cells exchange organic and inorganic molecules
that enable the growth and proliferation of both partners.
(Libudzisz et al., 2007)
 Factors influenced microbial growth in surface water
Grazer chain
Biotic factors
Example of realtionship between
organisms is a grazer chain.
Bacteria are component of grazer chain
in water environments.
Bacteria are decomposers. They
decompose dissolved organic matter
(DOM) - various organic substances to
simpler organic compunds).
They are consumed by eukaryotic
organisms (Protozoa) such as ciliates and
heterotrophic flagellates.
The numer of bacterial cells can be also
reduced by viruses – bacteriophages.
Viruses

(Libudzisz et al., 2007)

 Autochthonous microflora of surface water

The high diversity of aquatic environments and factors that act upon microorganisms
leads to high diversity of aquatic microorganisms.

Autochthonous microorganisms – natural, native microorganisms for some kind of

water environment e.g.:
 Purpure sulfur bacteria e.g. Chromatium okenii
 Sulfur-oxidizing bacteria e.g. Beggiatoa, Thiotrix
 Iron-oxidizing bacteria e.g. Leptothrix, Crenothrix, Gallionella,
 Chemoorganotrophs:Pseudomonas, Aeromonas, Vibrio, Spirillum, Spirochaeta
 Nitrifying bacteria: Nitrosomonas, Nitrobacter
 Cocci: Micrococcus, Sarcina
 Fungi: Mucor, Leptomitus

(Libudzisz et al., 2007)

 Chemoorganoheterotrophs - use organic compunds as a source of energy and carbon to
synthesize new organic compounds in their organisms. e.g. Pseudomonas, Aeromonas,
Vibrio, Spirillum, Spirochaeta

Cocci e.g.: Micrococcus, Sarcina

Environment: a wide range of environments, including water, dust, and soil; also in
environments with a low availability of water or high salt concentrations; aerobic
conditions.

Fungi: Mucor, Leptomitus

Leptomitus – filamentous water mould. It is saprophyte. It lives on detritus (dead remains
of plants and animals) and in waters contaminated with industrial sewage, which are rich in
organic matter.

Sulfur-oxidizing bacteria – e.g. Beggiatoa, Thiotrix.

Environment: aerobic, rich in hydrogen sulfide (H2S), including water that has been
contaminated with sewage. Because of this, the mats, that they form are good indicators of
water pollution.
They are chemolithoautotrophs. They oxidize sulfur compounds such as hydrogen
sulfide (H2S) and inorganic sulfur (S) to sulfuric acid (H2SO4).
Purpure sulfur bacteria
 Environment: anoxic, illuminated zones of lakes or other stagnant water bodies
with high concentration of hydrogen sulfide (H2S) .
 They are photoautotrophs. These bacteria are capable of photosynthesis. They
get energy from the oxydation of hydrogen sulfide (H2S) to elementar sulfur, e.g.
Chromatium okenii

Iron-oxidizing bacteria e.g. Leptothrix, Crenothrix, Gallionella

 Environment - near-neutral pH, low concentration of oxygen, and iron-rich
freshwaters and wetlands with low concentration of organic matter e.g. water
bodies consisted of groundwater extracted from mines.
 They are chemolithoautotrophs, they oxidize dissolved form of iron, ferrous ion
(Fe2+) to ferric (Fe3+) ion – brown sediment, insolube in water.

Nitrifying bacteria: Nitrosomonas, Nitrobacter.

• Environment: widely distributed in soil or water, where there are large amounts
of ammonia, such as lakes or streams into which treated and untreated sewage is
pumped; in aerobic environment.
• Chemolithoautothrophs; they oxidize ammonia (NH4) to nitrite (NO2-), and then
nitrite to nitrate (NO3-). This is nitrification process.
 Allochthonous microflora of surface waters

Allochthonous microflora – microorganisms which get into the surface water

from soil, air, plants, animals, people – especially with industrial and municipal
sewage.

They are mainly heterotrophs - saprophytes and pathogens:

 Bacteria living in sewage - rods: Pseudomonas, Proteus, Clostridium. They
tolerate high concentration of organic matter and low concentration of
oxygen.
 Faecal bacteria from animal’s and human faeces: Escherichia coli,
Enterococcus faecalis, Clostridium perfringens
 Pathogens from human and animals: Salmonella, Shigella, Vibrio cholerae,
Mycobacterium, Campylobacter and many others.

(Libudzisz et al., 2007)

 Bacterial diseases - examples
Pathogen Disease Environment, route of infection
Salmonella typhi Typhoid fever (pol. dur brzyszny): contaminated water and food, contact
(serovar) and high fever, weakness, abdominal with carrier of these bacteria
pain, constipation, headaches,
mild vomiting

Salmonellosis, sepsis
other salmonellae
Shigella dysenteriae Bacillary dysentery (pol. contact with ill people, insects,
czerwonaka bakteryjna): contaminated water and food
severe diarrhea and abdominal
pains
Vibrio cholerae O1 Cholera - profuse, watery oral route of infection, wastewater
diarrhea; vomiting; from household (domestic sewage)
rapid dehydration

Campyobacter coli Gastroenteritis (pol. nieżyt alimentary tract of wild animals,

C. jejuni żołądka i jelit) domestic and farm animals; meat
contaminated with faeces,
contaminated milk, water, infected
animals and people

(Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Bacterial diseases - examples
Pathogen Disease Environment, route of infection

Escherichia coli Gastroenteritis, Bloody diarrhea contaminated water and food

serovar O157 : H7 Hemolytic-uremic syndrome (pol.
zespoł hemolotyczno- mocznicowy):
anemia, kidney failure, low platelet
count

Legionella Legionellosis (pol. legioneloza): surface water, underground water,

Legionnaires' disease (pol. choroba thermal water, soil, water supply
legionistów), system, air conditioning equipment
Pontiac fever

Mycobacterium Tuberculosis (pol. gruźlica) water environment, sewage, water

tuberculosis supply systems, hot water systems,

(Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Viral diseases - examples
Pathogen Disease Environment, route of infection

Adenoviruses gastroenteritis, acute respiratory sewage, raw water sources and

diseases, pneumonia, diseases of treated drinking-water supplies
the urinary tract and the eyes

Astroviruses gastroenteritis, predominantly sewage, water sources and in

diarrhoea, mainly in children drinking-water supplies
under age of 5 years

Caliciviruses major cause of acute viral in domestic wastewaters as well as

(Noroviruses and gastroenteritis in all age groups. faecally contaminated food and
Sapoviruses) symptoms include nausea, vomiting water, including drinking-water
and abdominal cramps supplies.

Hepatitis E virus hepatitis E contaminated water and food

(Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Viral diseases - examples
Pathogen Disease Environment, route of infection
Enteroviruses one of the most common causes of human generally the most numerous in
infections; cause broad spectrum of diseases: sewage, water resources and
a mild illness with fever, myocarditis, treated drinking-water supplies
meningoencephalitis,, herpangina, neonatal
multi-organ failure, polymyositis, chronic
fatigue
Hepatitis A virus hepatitis A Person-to-person spread is probably
the most common route of
transmission, but contaminated
food and water are important
sources of infection
Rotaviruses the most important single cause of infant Person-to-person transmission,
death in the world; cause 50–60% of cases of the inhalation of aerosols; ingestion
acute gastroenteritis of hospitalized children; of contaminated food or water
watery diarrhoea with fever, abdominal pain
and vomiting, dehydration and metabolic
acidosis

(Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Protozoan diseases - examples
Pathogen Disease Environment, route of infection
Acanthamoeba culbertsoni Granulomatous amoebic many types of aquatic environments,
encephalitis (pol. ziarniakowe including surface water, tap water,
zapalenie mózgu) swimming pools and contact lens
solutions

Cryptosporidium Cryptosporidiosis (pol. contaminated water

hominis/parvum kryptosporidioza)
Cyclospora cayetansis Cyclosporiasis (pol. in sewage and water sources
cyklosporoza): watery
diarrhoea, abdominal
cramping, weight loss,
anorexia, myalgia, vomiting
and/or fever.
Entamoeba histolytica About 85–95% of human cysts may remain viable in suitable
infections are asymptomatic; aquatic environments for several
Acute intestinal amoebiasis months at low temperature. They are
(pol. pełzakowica), diarrhoea relatively resistant to disinfection and
with cramping, lower may not be inactivated by chlorination
abdominal pain, low-grade practices generally applied in the
fever production of drinking-water. The risk
for waterborne transmission is higher in
the tropics.
• Drinking-water contaminated with soil or faeces could act as a carrier of helminths.
However, in most of mentioned cases, the normal mode of transmission is ingestion of the
eggs in food contaminated with faeces or faecally contaminated soil rather than ingestion
of contaminated drinking-water
(Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)

Helminths = parasitic worms, are large parasites, which can generally be seen with the
naked eye. Helminths can be divided into three groups: cestodes (tapeworms),
nematodes (roundworms), and trematodes (flukes). The helminths differ from other
infectious organisms in that they have a complex body structure. They are multicellular and
have partial or complete organsystems (e.g., muscular, nervous, digestive, and
reproductive) (Encyclopedia Britannica).

Fluke Roundworm Tapeworm

Fasciola hepatica Ascaris lumbricoides Taenia solium
• Some microbs naturally present in the environment may be able to cause diseases
and be pathogens in vulnerable subpopulations:
• the elderly
• the very young,
• patients with burns or extensive wounds,
• patients undergoing immunosuppressive therapy
• people with acquired immunodeficiency syndrome (AIDS).
 If water used by such persons for drinking or bathing contains sufficient numbers of
these organisms, they can cause various infections of the skin and the mucous
membranes of the eye, ear, nose and throat.
 Examples of such agents are:
 Pseudomonas aeruginosa,
 Flavobacterium,
 Acinetobacter,
 Klebsiella,
 Serratia,
 Aeromonas
 certain “slowgrowing” (non-tuberculous) mycobacteria

(Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
Sources of fecal contamination

https://www.slideshare.net/fatooo1/microbial-source-tracking-markers-for-detection-of-fecal-contamination
 Classification of diseases related with water
• Water-borne (waterborne): an enteric microorganism enters the water via fecal
contamination and is transmitted through ingestion of the contaminated water. Examples
include typhoid and cholera.
• Water-washed: an enteric microorganism is transmitted through ingestion of fecal
material, but the presence of fecal material is due to a lack of water for bathing, washing of
hands, utensils, and so on. In these cases, the pathogen is not present in the water, but on
hands or utensils, and transferred to water, food, and so on that is then consumed.

• Water-based: a worm spends a part of its life cycle in the aquatic environment. Water-
based infections are of two types: those acquired via ingestion (such as Dranunculus
medinensis), and those acquired by contact with the water and penetration of worm
through the skin (such as with Schistosoma, the causative agent of schistosomiasis).

 Water-related: an insect that transmits the pathogen breeds in water. E.g. malaria.

 Water-aerosol: used for diseases, that are transmitted via aerosols such as legionellosis.

 „Engineered water system associated” – used for these disease agents that arise within
engineered systems (such as Legionella, which are naturally present in water but can
multiply to harmful levels in engineered water systems.” (Yates et al., 2016)
 Sanitary Analysis of Water – Indicator Microorganisms
Indicator microorganisms are used in sanitary water quality assessment to
estimate the possibility of water contamination of human and animal
pathogens (especially from fecal wastes).
If these bacteria are found in drinking water there is high probability that
pathogens are also in this water.
Criteria for indicator microorganism:
1. The indicator bacterium should be suitable for analysis of all types of water:
tap, river, ground, impounded, recreational, estuary, sea, waste.
2. The indicator bacterium should be present whenever enteric pathogenes are
present.
3. The indicator bacterium should survive longer than the hardiest enteric
pathogen.
4. The indicator bacterium should not reproduce in the contaminated water
and produce an inflated value.
5. The method of indicator bacteria analysis should be easy to perform.

(Prescott et al., 2004)

 Sanitary Analysis of Water – Indicator Microorganisms

6. The assay procedure for the indicator should have great specificity. It means
that other bacteria should not give positive results. The procedure should
have high sensitivity and detect low level of the indicator.
7. The indicator should be harmless to humans.
8. The level of indicator bacterium in contaminated water should have some
direct relationship to the degree of fecal pollution.
Indicator bacteria use in sanitary analysis of drinking water:
• Coliforms – pol. bakterie z grupy coli
• Escherichia coli – pol. pałeczka okrężnicy
• Enterococcus faecalis - pol. enterokok, paciorkowiec kałowy
• Clostridium perfringens – pol. laseczka zgorzeli gazowej

(Prescott et al., 2004)

 Sanitary Analysis of Water – Indicator Microorganisms
Coliform bacteria (latin colon – en. colon=large intestine – pol. jelito grube, okrężnica)

• Taxonomic position:
Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae

• Rod-shaped, non-sporing, Gram-negative, facultative anaerobic bacteria

• They ferment lactose, producing lactic acid and gas.
• Many members of this family are normal flora of the intestine of human and warm-
blooded animals.
• Some of them are also noted in soil (some Klebsiella species).
• Their concentration amounts: 106 – 109 coliforms per gram of feces.
• Main genera: Escherichia, Klebsiella, Enterobacter, Citrobacter.
 Sanitary Analysis of Water – Indicator Microorganisms

Coliforms (total coliforms – TC)

• These bacteria form red colonies on agar endo (type of selective medium) within 24h at
36ºC.
• Total coliforms include environmantal and human/animal types.

Fecal coliforms (FC)

• This group includes only bacteria coming from human/animal.
• They are grow on agar endo at 44.5ºC. In this temperature growth and activity of
environmental coliform-type bacteria are suppressed.

Coliforms bacteria on agar endo

(Maier et al., 2000; Prescott et al., 2004; Michałkiewicz and Fiszer, 2007;
Grabińska-Łoniewska and Siński, 2010; Sadowski and Whitman, 2011)
 Sanitary Analysis of Water – Indicator Microorganisms
Escherichia coli
• Bacterium belongs to normal flora of
mammals’ intestine.
• Noted also in sewage, natural waters, sand,
sediment, soil.
• Gram-negative
• Facultative anaerobe
• Cells are 2-3 µm long
• 4-8 polar flagella https://pixnio.com/science/microscopy-images/escherichia-coli/morphologic-details-
displayed-by-a-single-gram-negative-escherichia-coli-bacterium

• It is found in feces in amount 109 per gram

(~1% of the total bacterial biomass)
• It can survive in water at 15-18ºC for 4-12
weeks.

(Prescott et al., 2004; Michałkiewicz and Fiszer, 2007; Grabińska-Łoniewska and Siński, 2010; Sadowski and Whitman, 2011)
 Sanitary Analysis of Water – Indicator Microorganisms

Escherichia coli
• This bacterium grows on various selective media:
 m-Endo Agar

Red colonies with a

metallic sheen
 Sanitary Analysis of Water – Indicator Microorganisms

Escherichia coli
• This bacterium grows on different selective media:
 Lactose TTC Agar with Tergitol™-7

colonies in yellow, orange and

red with a yellow central zone
under the filter
 Sanitary Analysis of Water – Indicator Microorganisms

Escherichia coli
• This bacterium grows on different selective media:
 Chromogenic Coliform Agar (CCA) – blue-violet colonies
Sanitary Analysis of Water – Indicator Microorganisms

+ Fecal Contamination

Pathogenic strains of Escherichia coli (red star)

 Sanitary Analysis of Water – Indicator Microorganisms
Enterococcus faecalis
• Taxonomic position Domain: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Enterococcaceae
Genus: Enterococcus
https://pixnio.com/?s=enterococci

• Gram-positive, facultative anaerobes, nonsporing bacteria that occur as cocci,

diplococci or streptococci.
• Commensal in the human and warm-blooded animal intestine.
• It occurs in the human colon in amount of 108 CFU per gram feces (~1% of the total
bacterial biomass)
• !!! Most strains of Enterococci can survive longer in the water than coliform bacteria.
• !!! Some enterococci are more resistant to chlorine than coliforms.

(Prescott et al., 2004; Michałkiewicz and Fiszer, 2007; Grabińska-Łoniewska and Siński, 2010; Sadowski and Whitman, 2011)
 Sanitary Analysis of Water – Indicator Microorganisms

Enterococcus faecalis
This bacterium is detected on selective medium:
• Slanetz and Bartley Agar – red-brown colonies
 Sanitary Analysis of Water – Indicator Microorganisms
Clostridium perfringens
• Taxonomic position Domain: Bacteria
Phylum: Firmicutes
Class: Clostridia
Order: Clostridiales
Family: Clostridiaceae https://pixnio.com/science/microscopy-images/clostridium-perfringens/photomicrograph-
reveals-clostridium-perfringens-grown-in-schaedlers-broth-using-gram-stain
Genus: Clostridium

• Gram-positive, obligate anaerobic, non-motile, rod-shaped

• Produces endospores
• Noted in soil, in feces, sewage
• Sulfates reducing, fermentative bacterium
• Commensal organisms for human and animals but can cause different diseases e.g.
gangrene
• It is useful indicator of long-term water contamination water because of spore
production.
• Its detection indicates past or current fecal contamination depending on the ratio of
spores to vegetative cells.
RATIO spores : vegetative cells
the more spores is noted in the water – the elder water contamination is
(Prescott et al., 2004; Michałkiewicz and Fiszer, 2007; Grabińska-Łoniewska and Siński, 2010; Sadowski and Whitman, 2011)
 Sanitary Analysis of Water – Indicator Microorganisms

Clostridium perfringens
This bacterium is detected on selective medium:
• m-CP agar – colonies that change their color from yellow to pink after put them under
the influence of ammonium hydroxide vapors
 Quality of water intended for human consumption
International Guidelines and Law Regulations
• Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for
human consumption (OJ L 330, 5.12.1998, p. 32)
(Dyrektywa Rady 98/83/WE w sprawie jakości wody przeznaczonej do spożycia przez
ludzi)
• This Directive has been changed in 2015 by the commission directive:
Commission Directive (EU) 2015/1787 of 6 October 2015 amending Annexes II and III to
Council Directive 98/83/EC on the quality of water intended for human consumption
(Dyrektywa Komisji (UE) 2015/1787 z dnia 6 października 2015 r. zmieniającej załączniki II
oraz III do dyrektywy Rady 98/83/WE w sprawie jakości wody przeznaczonej do spożycia
przez ludzi)
 Sanitary Analysis of Drinking Water – Polish Law Regulations

In Poland , they are few law regulations related to drinking water quality.
The main acts are:

• The Act of June 7, 2001 on collective water supply and collective sewage disposal
[Dz.U. 2001 poz. 747]
(Ustawa z dnia 7 czerwca 2001 r. o zbiorowym zaopatrzeniu w wodę i zbiorowym
odprowadzaniu ścieków [Dz.U. 2001 poz. 747])

• Regulation of the Minister of Health of 7 December 2017. On the quality of water

intended for human consumption [Dz.U. 2017 poz. 2294]
(Rozporządzenie Ministra Zdrowia z dnia 7 grudnia 2017 r. w sprawie jakości wody
przeznaczonej do spożycia przez ludzi [Dz.U. 2017 poz. 2294])
This regulation contains requirements presented in the Council Directive.
 Sanitary Analysis of Drinking Water – Polish Law Regulations

Regulation of the Minister of Health of 7 December 2017. On the quality of water

intended for human consumption [Dz.U. 2017 poz. 2294]
The regulation specifies (§1):
1) quality requirements of water intended for human consumption, including
bacteriological, physico-chemical and organoleptic parameters;
2) the method of assessing the suitability of water;
3) the minimum frequency and sampling points of water;
4) the scope of water quality testing;
5) water quality monitoring program;
6) the method of supervision over materials and products used in the processes of water
treatment and distribution;
7) the method of supervision over laboratories performing water quality tests;
8) the way of informing consumers about the water quality;
9) the way of proceeding towards the authorities of the Sanitary Inspectorate in case of
not meeting the water quality requirements.
 Sanitary Analysis of Drinking Water – Polish Law Regulations
The provisions of the Regulation shall not apply to (§2):
1) natural mineral waters that meet the requirements specified in the Act of 25 August 2006
on food and nutrition safety (Journal of Laws of 2017, item 149 and 60);
2) spring waters and table waters in the scope regulated in the regulations issued on the basis
of art. 39 of the Act of August 25, 2006 on food and nutrition safety;
3) healing waters:
a) indicated in art. 5 par. 2 point 1 of the Act of 9 June 2011 - Geological and Mining Law
(Journal of Laws of 2017, item 2126),
b) for which sanitary conditions and requirements are specified in the regulations issued on
the basis of art. 36 par. 5 of the Act of 28 July 2005 on spa treatment, health resorts and areas
of spa protection as well as on spa communes (Journal of Laws of 2017 item 1056);
4) water originating from individual water intakes supplying less than 50 people or providing
less than an average of 10 m3 of water per day,
unless water is supplied as
• part of a business
• or for public buildings
• or for buildings of collective residence (e.g. hotel, motel, prison, orphanage, pensioners’s home)
• or for food business.
 Sanitary Analysis of Drinking Water – Polish Law Regulations
In § 3. 1-2.:
The water is safe to use if it is free of:
 pathogenic microorganisms and parasites in a number that causes a potential threat to
human health,
 all substances in concentrations that are potential threat to human health
 does not show corrosive properties
 and meets the requirements:
1) microbiological - in Part A of Annex 1 to the Regulation;
2) chemical - specified in Part B of Annex No. 1 to the Regulation.

Domestic hot water should, in addition to the above-mentioned requirements meet the
requirements mentioned in Part A of Annex 5 to the Regulation.
 Sanitary Analysis of Drinking Water – Polish Law Regulations
§ 3.3.:
The water and sewerage companies and entities providing or using water from an
individual intake as part of an economic activity or in public buildings, buildings of a
collective residence, or in food business
have to take all measures to ensure that the water meets the requirements specified for:
1) the indicator parameters specified in Part C of Annex No. 1 to the Regulation;
2) additional chemical requirements set out in Part D of Annex No. 1 to the Regulation;
3) radioactive substances specified in Annex 4 to the Regulation.
 Sanitary Analysis of Drinking Water – Polish Law Regulations
Regulation of the Minister of Health of 7 December 2017. On the quality of water intended
for human consumption [Dz.U. 2017 poz. 2294, Annex 1, Part A, C]
Rozporządzenie Ministra Zdrowia z dnia 7 grudnia 2017 r. w sprawie jakości wody
przeznaczonej do spożycia przez ludzi [Dz.U. 2017 poz. 2294, Załącznik Nr 1 cz. A, C]
Annex 1 Part A Microbiological parameters
Table 1. Microbiological requirements
Parametric value

Parameter number of
microorganisms [CFU or sample volume [ml]
MPN]

Escherichia coli 0 100

Enterococci 0 100

CFU- colony forming unit

MPN – most probable number of bacteria
 Sanitary Analysis of Drinking Water – Polish Law Regulations
Annex 1 Part C Indicator parameters
Table 4. Microbiological requirements
Parametric value

Parameter number of Explanations

sample volume
microorganisms
[ml]
[CFU or MPN]
Coliforms 0 100 1)

The total number of without abnormal 2)

microorganisms at 22±2ºC changes
Clostridium perfringens (with
0 100 3)
spores)

Explanations:
1)
Individual bacteria <10 cfu (NPL) are allowed. If coliform bacteria are detected <10 CFU (NPL) / 100 ml, the E. coli and
enterococcus parameter should be examined in connection with § 21 para. 4 of the Regulation.
2
) It is recommended that the total number of microorganisms should not exceed:
- 100 cfu / 1 ml in water entering the water supply system,
- 200 cfu / 1 ml at consumer tap.
3)
It should be tested in water originating from surface and mixed intakes, and in the case of exceedance limit values,
the water should be tested to ensure that there is no risk to human health arising from presence of other pathogenic
microorganisms, e.g. Cryptosporidium.
 Sanitary Analysis of Drinking Water – Polish Law Regulations
Cryptosporidium
• Single-celled, obligatory intestinal parasitic protist
• Parasite of mammals, birds, reptiles, fishes
C. parvum - ruminants, dogs, cats, rabbits, rodents, people
C. hominis - people and monkeys
• Oocysts are commonly present in natural waters and soil, also
in food and surfaces contaminated with excrement of animals
and infected people; in drinking-water supplies. https://pixnio.com/science/microscopy-images/cryptosporidiosis-
cryptosporidium/stool-smear-micrograph-revealing-cryptosporidium-
• Thick-walled oocysts with a diameter of 4–6 μm are shed in parvum-as-the-cause-of-this-patients-cryptosporidiosis

faeces,
• Cryptosporidium
In raw sewage: 14 000 oocysts per liter
In surface waters 5 800 oocysts per liter
• Humans and livestock, particularly young animals,
are the most significant source of infections.
Calves can excrete 1010
oocysts per day. Infected
people 10 oocysts per day (also 1-4 week after
12

disappearance of disease symptoms)

•Oocysts can survive for weeks to months in fresh water.
•They can live in the wide range of temperature -20 - 60ºC
(Matuszewska, 2007; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
https://hoards.com/article-6433-cryptosporidium-parvum-is-silently-stealing-pounds--and-threatening-your-family.html
 Sanitary Analysis of Drinking Water – Polish Law Regulations

• Human health effects – cryptosporidiosis :

 Self-limiting diarrhea, sometimes nausea, vomiting and fever, which usually
resolves within a week in normally healthy people (but can last for a month or
more).
 Infections in immunocompromised people can be life-threatening.
• Ingestion of less than 10 oocysts can lead to infection.
• Cryptosporidium is transmitted by the fecal–oral route.
• The major route of infection is person-to-person contact.
• Other sources of infection: consumption of contaminated food or water, direct contact
with infected farm animals and domestic pets.
• Outbreaks have been reported in hospitals, day-care centers, within households, among
bathers (affecting participants in water sports in lakes and swimming pools), and in
municipalities with contaminated public water supplies.
• Water distribution systems are particularly vulnerable to contamination with
Cryptosporidium, which can survive most of disinfection procedures such as
chlorination. More effective is ozone as disinfectant and UV light treatment.

(Matuszewska, 2007; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Sanitary Analysis of Drinking Water – Polish Law Regulations
Annex 5 Microbial requirements that should be met by hot water, minimum frequency of
hot water sample collection and procedures depending on the results of bacteriological
testing

Part A Microbial requirements that should be met by hot water.

Parametric value

Parameter number of Explanations

sample volume
microorganisms
[ml]
[CFU]
<100 100 1)
Legionella sp.
<50 1000 2)

Explanations:
1)
It should be examined in samples of hot water collected in enterprises of the entity performing
medical activities such as stationary and round-the-clock health services and in buildings of collective
residence and in public buildings, in which water and air aerosol are produced during their use.
2)
The value of the parameter applies to enterprises of the entity performing medical activities, such as
stationary and round-the-clock health services, in which patients with immunodeficiency reside,
including those treated by immunosuppressive treatment.
 Sanitary Analysis of Drinking Water – Polish Law Regulations
Legionella pneumophila
• Taxonomic position
Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Legionellales
Family: Legionellaceae https://pl.m.wikipedia.org/wiki/
Plik:Legionella_pneumophila_(SEM).jpg
Genus: Legionella

• Gram-negative, non-spore-forming, rod-shaped, aerobic bacterium,

• Free-living bacteria are 0.3-0.9 µm wide and ~1.3 µm long, in vitro they can grow to
2-6 µm and form filaments 20 µm long.
• They are motile – one or more polar or subpolar flagellae.
• The optimal temperature of growth in-vitro is 36ºC (range is 15-43ºC).
• Generation time is 99 minutes under optimal conditions.
• The natural habitat is fresh water and soil. Bacteria live in the cell of other
microorganisms – natural hosts are other bacteria or Protozoa e.g. amoebae. They
are relatively slow-growing, ubiquitous, aquatic saprophytes.

(Stypułkowska-Misiurewicz and Pancer, 2002; Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth
edition incorporating the first addendum. WHO; 2017)
 Legionella pneumophila
• Legionella has been noted mainly in humans (rarely in animals: guinea-pigs, rats,
mice, monkeys)
• Their reservoir of bacteria are various man-made hot-water systems such as:
• water cooling systems,
• cooling towers,
• swimming pools,
• jacuzzi,
• air conditioning systems,
• It can grow in all types of devices producing droplets of water:
• showerheads and sinks,
• fountains,
• garden sprinklers,
• car washes,
• dental office water e.g. used for dental turbines
• hot springs.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Legionella pneumophila

• Their growth in water installation is favored by:

 Water stagnation,
 Sediment formation
 Temperature within 20-50°C, (mainly 35-45°C) – hot water systems
 The presence of biofilm – formed by microorganisms living inside the pipes and
reservoirs - This is an= kind of mircobial ecosystem allowing the Legionella to
multiply into a concentration that can be harmful for human health or life.

• The special type of risk are water systems in hotels, sanatoriums, retirement homes,
hospitals, sports venues.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Legionella pneumophila - Pathogenicity for humans
• In 1976 Legionella was first detected as human pathogen after outbreak of pneumonia
in a hotel in the United States (Philadelphia).
• It was during veterans of the United States (called American Legion) meeting.
• Bacteria grew and spread in the hotel's hot water installation.
• As a result of the disease:
 221 people (guests and hotel staff) became ill with a disease similar to
pneumonia.
 34 people died
After histopathological examination it was discovered that Legionella caused diseases and
death of veterans and hotel staff.
The name of bacterium – Legionella and the name of disease – Legionnaires’ disease
come form the name of organization associating veterans of US Marines – American
Legion.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Legionellosis – infection route
• Legionella pneumophila is relative intracellular pathogen.
• In the environment Legionella is able to enter a viable but non-culturable (VBNC) stage
- this is a dormant stage.
• The bacteria can also live in biofilms and grow within Protozoa cells (amoeba cells).

• During infection bacteria get f.e. with the aerosol to the host's alveoli where are
phagocytized by macrophages.
• They can survive inside the cells of macropages. They replicate and breakdown tchem,
what leads to spreading of infection. Spreading occurs through the bloodstream and
lymphatic system.

• The genus Legionella includes more than 48 species. The species L. pneumophila is
responsible for more than 98% of the Legionnaires disease cases, other species such as
L. anisa, L. micdadei, L. dumoffii or L. feeleii are rarely pathogenic in humans, although
they relatively frequently colonize water distribution systems.
• Most susceptible to legionellosis are elderly, smokers, diabetes and
immunocompromised persons.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Sanitary Analysis of Drinking Water – Polish Law Regulations
Legionella pneumophila - Pathogenicity for humans
• Legionellosis is the name used for all types of infections caused by Legionella.
• According to the International Classification of Diseases and Health Problems adopted by
Poland legionellosis should be registered depending on disease form as: A48.1
legionnaires‘ disease or A48.2 legionnaires' disease without symptoms of pneumonia
(Pontiac fever). Also extrapulmonary forms of legionellosis are observed.
A48.1 Legionnaires' disease
• This is the type of a heavy, often bilateral pneumonia.
• The incubation period for the infection is between 2 to 10 days.
• The course can be varied - from the uncomplicated form of inflammation of lungs for
severe respiratory failure
• The disease starts suddenly, in the form of non-specific symptoms such as headaches
and muscles ache, lack of appetite, bad mood, general weakness. Then a high fever
comes in (above 40ºC) with chills and a dry cough. Sometimes cough with blood,
shortness of breath, chest pain and difficulties in breathing.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Sanitary Analysis of Drinking Water – Polish Law Regulations

A48.1 Legionnaires' disease

• Other symptoms – non typical for pneumonia occurs in some patients:
 on the part of the digestive system: nausea, vomiting, diarrhea, abdominal pain;
 from the nervous system: headaches, ataxia, stiff neck, excessive drowsiness,
disturbances of consciousness, confusion, dementia syndrome, hallucinations.
• 1-5% of Legionella infections cause Legionnaries’ disease.
• The mortality among patients ranges from 15% to 20%, and in hospitalized patients
with compromised immune systems more than 30%.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Sanitary Analysis of Drinking Water – Polish Law Regulations

A48.2 Pontiac fever

• It is a mild form of infection with Legionella pneumophila (occurs in more than 90% of
the exposed population)
• It occurs much more frequently than the pulmonary type.
• The incubation period is from 24 to 48 hours.
• Risk of infection it is high, it usually spreads epidemically among adults and children.
• The symptoms are: a sudden onset with a fever and flu-like symptoms: bad mood,
muscular pains, headaches, fatigue, weakness. Next symptoms are: sore throat, runny
nose, unproductive cough. Sometimes observed: nausea, vomiting, diarrhea, dizziness.
• Symptoms of disease disappear spontaneously within a few days.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Sanitary Analysis of Drinking Water – Polish Law Regulations

Extrapulmonary forms of legionellosis

• They occur very rarely, but have a very severe course.
• Bacteria can cause heart diseases e.g. myocarditis, pericardium and endocardium.
• Other diseases are eg.: acute kidney failure, paranasal sinusitis, connective tissue
inflammation, postoperative wounds infection.

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 Sanitary Analysis of Drinking Water – Polish Law Regulations

How to prevent Legionella in hot water system?

• Minimise water stagnation in water system

• Avoid water temperatures optimum for Legionella growth (30-45°C).

• The water temperature in hot water system should be ≥ 50°C

• Eliminate scale, sediment and biofilm

• Eliminate of blind sections of the installation

• Prevent corrosion in water system

• Systematically carry out cleaning and disinfection

• Replace the oldest sections of the installation (the older the installation is, the higher
the probability of Legionella is)
 Sanitary Analysis of Drinking Water – Polish Law Regulations

Methods of disinfection hot water system:

• Thermal disinfection (superheat and flush) – is based on heating the water to the
temparature >60°C in water heater and flushing the hot water system by opening all
taps.
 If the temperature is 70°C – the required time is 5 min
 65°C – the required time is 10 min
 60°C – the required time is 30 min

• Chemical disinfection – hyperchlorination, ozonation, treating with chlorine dioxide,

ionization with silver and copper

• Ultraviolet rays

(Steinert et al., 2002; Grabińska-Łoniewska and Siński, 2010; Guidelines for drinking-water quality: fourth edition incorporating the first addendum. WHO; 2017)
 References
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chorobotwórcze w ekosystemach wodnych i sieciach wodociągowych. Wydawnictwo „Seidel-
Przywecki” Sp. z o.o., Warszawa.
• Guidelines for drinking-water quality: fourth edition incorporating the first addendum. Geneva:
World Health Organization; 2017.
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środowiska ich występowania. Tom 1., PWN, Warszawa.
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Occurence in water environment and health risk. ROCZN. PZH 58, NR 3, 569-577.
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Washington, DC.
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• Yates M.V., Nakatsu C.H.,. Miller R.V., Pillai S.D. (eds.) 2016. Manual of Environmental
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