Lecture on bacteriological risk in drinking water for the faculty of Veterinary Medicine, University of Helsinki; Course in Environmental Health

1. BACTERIA IN DRINKING WATER Talousvesi
Talousvesi (763/1994) 16 §
…
1) kaikkea vettä, joka on tarkoitettu juomavedeksi, ruoan
valmistukseen tai muihin kotitaloustarkoituksiin riippumatta
siitä, toimitetaanko vesi jakeluverkon kautta, tankeissa,
pulloissa tai säiliöissä; sekä
2) kaikkea vettä, jota elintarvikealan yrityksessä käytetään
elintarvikkeiden valmistukseen, jalostukseen, säilytykseen ja
markkinoille saattamiseen

2. LEARNING OUTCOMES
Outline factors affecting the composition of (normal) DW microbioma
List and categorize bacterial waterborne pathogens transmitted through DW
Classify risk factors for the presence of bacterial pathogens in DW and the
transmission to human
Review possible epidemiological scenarios of waterborne bacterial infections
transmitted through DW

3. THE ”NORMAL” FLORA In drinking water
Which bacteria are present in the tap water?
Are they dangerous?
From where they come?

4. WHAT DO WE DRINK FROM THE TAP?
Processes used for producing potable water are not intend to
produce bacteria-free water
Rather they are concerned with removing hazardous bacterial
species and making water aesthetically pleasing
Little is known about bacterial composition that is dispensed from
point-of-use taps in water distribution systems

5. WHAT DO WE DRINK FROM THE TAP?
Bacterial concentrations in public drinking water distribution systems
are estimated ~ 106 - 108 cells/L (planktonic phase)*
~ 250 000 – 25 000 000
~ 25 – 2 500
*representing less than 2% of bacteria in a distribution network
Lautenschlager et al. Water. Res. 2013; Liu et al. Biomed. Res. Int. 2013; Nescerecka et al. PLOS ONE. 2014; Liu et al. Environ. Sci. Technol. 2014

6. THE CULTIVABLE FRACTION OF PLANKTONIC
PHASE
Only a small fraction (~0,01%) is cultivable heterotrophic bacteria$:
Acinetobacter spp. *, Aeromonas spp.*, Alcaligenes spp., Comamonas spp., Enterobacter spp.,
Flavobacterium spp., Klebsiella spp.*, Moraxella spp. *, Pseudomonas spp.*, Legionella spp.*,
Sphingomonas spp., Stenotrophomonas spp., nontuberculous mycobacteria*, Bacillus spp.,
Nocardia spp.
*Specific strains of species detected in potable water can cause infection in certain vulnerable
people and, in favorable conditions, be responsible of community or nosocomial outbreaks
The cultivable species predominant in households, particularly in warm-water
distribution systems are Legionella spp. and Pseudomonas spp.
$Heterotrophic are bacteria that required organic carbon to growth

7. PLANKTONIC PHASE AT POINT-OF-USE IS
DOMINATED BY PROTEOBACTERIA*
Prest et al. Water Res. 2014
Proteobacteria
*similar results by: Pinto et al. Environ. Sci. Technol. 2012; Lautenschlager et al. Water Res. 2014;

8. DRINKING WATER MICROBIOME IS DYNAMIC
Shift in concentration, pattern and community structure between water treatment plan
and distribution network, and during retention time in the network
Prest et al. Water Res. 2014 Lautenschlager et al. Water. Res. 2013
”regrowth”

9. BIOFILM IN DRINKING WATER DISTRIBUTION
SYSTEMS
From: Proctor and Hammes. Current Opinion in Biotechnology, 2015

10. MICROBIOME CONTINUUM:
FROM SOURCE TO HOUSEHOLDS
From Proctor and Hammes. Current Opinion in Biotechnology, 2015
Major effects
on microbial
quantity and
composition

11. DRINKING WATER MICROBIOME
Highly diverse drinking water
microbiomes comprise up to 40 phyla
Differs across phases (planktonic,
biofilms, and loose deposits),
geographical location, type of source
Dynamically changes through stages
of treatment and distribution
Comprehensive definition of the
‘core’ microbiome is missing
From Proctor and Hammes. Current Opinion in Biotechnology, 2015

12. WATERBORNE INFECTIONS bacteria
Which bacterial pathogens are transmitted to human
through drinking water? Which diseases do they cause?
In which epidemiological contest?

13. WATERBORNE INFECTIONS: WHEN DO THEY
HAPPEN?
The introduction in the water
system of hazardous bacterial
species (usually fecal-associates)
as contaminants
Regrowth in the network (premise
water pipes) of opportunistic
bacteria
Increased host susceptibility - HIV
patients, ICU (nosocomial
outbreaks) – to normal water flora
Disease
Infection agent
Host susceptibilityEnvironment
• Dispersion
• Infection dose
• Survival/Duplication
• Virulence
• Route of transmission
• Immunological status
• Genetics
• Predisposing clinical conditions
• Risk status (pregnant, children,
elderly)
• Climate and weather
conditions
• Human and animal
density

14. ACCESS TO CLEAN WATER IS A CRITICAL FACTOR

15. FECAL-ASSOCIATED BACTERIAL
PATHOGENS
Diarrhea causing agents

16. FECAL-ASSOCIATED WATERBORNE BACTERIAL
PATHOGENS
Are bacteria in human or animal waste that entered water as
contaminants
Ingestion of drinking water as main route of infection – fecal-oral
route
Are not normal “inhabitants” of water:
- Highly virulent species or strains
- Low infection dose
- Healthy people are usually susceptible to the infection
Generally are not capable of growing in the water supply

17. HOW FECAL-ASSOCIATED BACTERIA ENTER THE
SYSTEM
Fecal contamination of ground
water by surface water, without
subsequent appropriate
disinfection
Contamination of distribution
systems through:
- Cross-connections
- Back siphonage
- Main breaks or repairs
- Inadequately protected storage
tanks/towers
- Natural disasters
Surface waterWastewater
Floods, landslip, …Main repair Main breaks
Surface water

18. DISPERSION AND PERSISTENCE IN THE WATER
ENVIRONMENT
Human or animal population density
Pathogen prevalence in the host populations
Shedding patterns – number of bacteria excreted and time of
shedding
Resistance to stress - pH, temperature, desiccation, ionization,
radiation (UV), antimicrobial agents, starvation
Resistance to biological predation - Phage, Protozoa
Stable environmental status - biofilm, spore, persister, filamentous
cell, dormancy
High Dispersion
Good Survival
or Adaptation

19. FECAL-ASSOCIATED WATERBORNE BACTERIAL
PATHOGENS AND DW-BORNE OUTBREAKS
Fecal waterborne bacterial pathogens are frequently associated with DW
outbreaks
A large proportion of the outbreaks affected a low number of peoples and is
linked to single-household water supplies à well-water is not chlorinated
Most outbreaks in Nordic countries are linked to private groundwater
- cross-connection with wastewater network
- contamination of well water by surface water

20. WATERBORNE OUTBREAKS IN FINLAND
59 waterborne outbreaks from 1998 – 2012
(3,9/y)
- Total of 22 594 people involved
Largest proportion of the outbreaks exposed
and infected people were 10 to 50
The biggest outbreak reported was Nokia
2007
- 1222 subjects (C. jejuni, norovirus, Giardia, Salmonella) à
cross-connection between the waste water system and
drinking water network
0
5
10
15
20
25
30
35
40
< 10 10-50 50-100 100-500 500-1000 1000-5000 >5000
Exsposed Infected
N.outbreaks
Norovirus
Campylobacter
Unknown cause
Chemical
Salmonella
Rotavirus
Giardia/Crypto.
Adenovirus
Astrovirus
Enterovirus
Norovirus 37%
Unknown 30%
Campy 19%

21. SEASONALITY WATERBORNE OUTBREAKS IN
FINLAND
1998 – 2012 Guzman-Herrador et al., 2015
It is particularly evident for Campylobacter

22. FECAL-ASSOCIATED BACTERIAL PATHOGENS
TRANSMISSIBLE THROUGH DW
-Campylobacter jejuni (coli)
-Pathogenic E. coli
-Yersinia enterocolitica
-Yersinia pseudotuberculosis
-Salmonella enterica
-Shigella spp.
THL surveillance information

23. CAMPYLOBACTER IS THE NUMBER ONE BACTERIAL
WATERBORNE PATHOGEN IN FINLAND
The most commonly reported gastrointestinal zoonotic bacterial
pathogen in humans in EU – estimated true incidence 2-5 mil
infections /year; C. jejuni – 90-95% infections
The estimated cost (public health systems and lost productivity) in
EU is EUR 2 400 million/year
Acute self-limiting enteritis and severe post-infections sequelae
Infections are generally sporadic. When outbreaks occurs they are
usually associated to consumption of contaminated water or raw
milk

24. SEASONALITY OF CAMPYLOBACTERIOSIS
- The transmission of Campylobacter
to human is a complex ecological
process with multiple hosts and
routes
- Short-term increase in temperature
is not associated with increased
transmission (unlike Salmonella)
- Intra-annual changes in the animal
host reservoir may be an important
explanation for the seasonal
patterns
August
Water
Decrease
detection of
Campylobacter in
water during (hot)
summer times
July

25. CAMPYLOBACTER IS (GENERALLY) MORE SENSITIVE
THAN E. COLI AND SALMONELLA TO PHYSICAL STRESS
Inactivation of C. jejuni in river water (dark): inoculum 107
Inactivation of C. jejuni in well water (dark): inoculum 105
Rodriguez and Araujo, J. Water. Health. 2012
González and Hänninen, J. Appl. Microb. 2012
4°C
4°C25°C
25°C
Natural inactivation of C. jejuni in in situ experiment in
river water: inoculum 107
Rodriguez and Araujo, J. Water. Health. 2012
---- Sun light experiments
Shade experiments

26. BIOFILM FORMATION AND INTERACTION WITH
FREE-LIVING PROTOZOA
C. jejuni in monoculture have been shown to
attach to an abiotic surface and form
biofilms to various degrees enhancing its
survivability in the environment
Protozoa in drinking water systems can delay the
decline of C. jejuni viability and increase C. jejuni
disinfection resistance
Martin Kalmokoff et al. J.
Bacteriol. 2006
C. jejuni 11168 attached to
various surfaces. (A) Stainless
steel, (B - C) nitrocellulose. (D
- E) glass fiber filters. (F) flhA
mutant demonstrating a loss in
the ability to attach to the glass
fiber filter, in comparison to the
image in panel E. Bars, 10 μm Snelling et al. Appl. Environ. Microbiol. 2005
(A and B) Microscopy of A. castellanii (CCAP 1501/10) after 3 days of
coculture with C. jejuni NCTC 11351 in PAS at 25°C. C. jejuni was stained
with Baclight viability dye before coculture (1:1) with A. castellanii.
magnification, ×40. The arrows indicate A. castellanii vacuoles containing
dead C. jejuni

27. STABLE ENVIRONMENTAL STATUS OF C. JEJUNI:
FILAMENTOUS CELL
37°C 4°C
--- filamentous forms Ghaffar et al. Front. Microbiol. 2015
• Filamentation has been identified in many
different bacteria and is thought to occur
through inhibition of cell division, metabolic
changes, or DNA damage
• Filamentation occurred spontaneously on entry
in to stationary phase
• It has been associated with stress and
starvation conditions during which it may
confer survival advantages à higher
survival in water

28. HOW CAN CAMPYLOBACTER BE SUCH A
SUCCESSFUL AS WATERBORNE PATHOGEN?
C. jejuni is tremendously successful in competing with the human
intestinal microbiota
Low infectious dose: few hundreds bacteria is sufficient to
overcome the colonization resistance of humans and can lead to
campylobacteriosis
C. jejuni has an enormous population size due to the colonization
of several animal hosts (many of them being livestock species)

29. MULTI-HOSTS à HIGHER DISPERSION
EHEC Campylobacter
≥ 104 cfu/g feces
< 104 cfu/g feces
1010 cfu/g feces

30. IMPACT OF LIVESTOCK ON ENVIRONMENT DISPERTION
1010 cfu/g feces
140
3500
0
5 Mil ~20 kT feces/month
5% Campylobacter positive flocks in summer time
= 1 kTons of feces/month à 10 000 000 000 000 000 000 of Campylobacter cells/month
= 2,5 X
How much fecal material do broiler chickens
produce for every cycle (~30 days) in Finland?
Campylobacter
Lowest known infectious dose is 800 cells

31. SOURCE OF WATER CONTAMINATION
BY C. JEJUNI
Example from Luxemburg and The Netherland
(Mughini-Gras 2016)
Water contamination mainly by one type (ST-45cc)
Luxemburg à Wild birds (chicken quite relevant)
The Netherlands à Chicken
ST-45 ST-45

32. OPPORTUNISTIC PREMISE PLUMBING
PATHOGENS
Acute febrile respiratory illness

33. OPPORTUNISTIC PREMISE PLUMBING PATHOGENS
(OPPPS)
Specific strains of species detected in potable water can cause infection in certain vulnerable
people and, in favorable conditions, be responsible of community or nosocomial outbreaks
-Legionella spp.
-Atypical mycobacterium
(Mycobacterium avium complex)
-Pseudomonas aeruginosa
-Aeromonas hydrophila

34. COMMON FEATURES OF OPPPS
Disinfectant resistance
- Chlorine or chloramine concentration required to kill 99.9% of the bacterial
OPPPs is much higher than usually applied
- The presence of residual disinfectant provides these resistant OPPPs with a
competitive advantage
Biofilm formation
- Increased resistance to disinfections
- Growth in biofilm in water pipes
- Makes bacterial more accessible to free-living, phagocytic amoebae
(enhance the proliferation in water)

35. COMMON FEATURES OF OPPPS
Survival at high temperatures
- High temperatures that are encountered in hot water pipes (35-45°C)
stimulate growth
Growth in free-living phagocytic amoebae
- OPPPs are not necessarily killed by amoebae following phagocytosis but can
actually survive and grow

36. COMMON FEATURES OF OPPPS
Virulence vary between strains with different infectious doses
Infectious dose usually high for healthy people but decrease
significantly in case of presence of predisposing diseases
Higher risk for immunocompromised persons (HIV, ICU, etc.)
Several clinical presentations à the most frequent is acute febrile
respiratory illness (dermatitis, lymphadenopathies)
Inhalation and contact as preliminary infection routes

37. POSSIBLE MEASURES TO REDUCE OPPP NUMBERS
IN PREMISE PLUMBING
Falkinham et al., Environ. Health Prot. 2015

38. LEGIONELLA Biology, epidemiology and
control

39. LEGIONELLA
A thin and flagellated gram-negative non-capsulated rod-like bacteria
The genus comprises 50 species and 70 distinct serogroups
20-30% of cases: other Lpn serogroups
L. pneumophila (Lpn) serogroup 1 most common agents of Legionnaires’ Disease (70%
of identified cases)
5-10% of cases: other non-pneumophila (L. micdadei – 60%; L. bozemanii – 15%;
others – 25%)
Pathogenesis is mainly due to the ability of L. pneumophila to invade and multiply
within human macrophages
https://legionnaires.ecdc.europa.eu/

40. CLINICAL PRESENTATIONS
Legionnaires’ disease is an atypical
pneumonia that might clinically
resemble pneumococcal or other
bacterial pneumonias
Symptoms range from mild disease
to severe pneumonia requiring
hospital admission

41. BULK AQUEOUS ENVIRONMENTS
The main reservoir is tap water system
Temperature
- Legionella multiply at temperatures in the range 25 - 42ºC, with an optimal temperature for
growth of 35ºC
- Legionella are killed when exposed to higher temperatures, in the range 55 to 65ºC
pH
- Good survival in lower pH values in the range 4 – 7 (2 log drop in viable count over a
month in tap water)
- Reduced survival at higher pH values (pH 8; 6 log drop in viable count over a month in tap
water)

42. AIRBORNE INFECTIONS
Main route à the inhalation aerosolised Legionella bacteria
The viability of aerosolised organisms decreases over time
Desiccation, increasing concentrations of solutes originating from the bulk liquid
Humidity, survival improved relative humidity reached 60%; positive association with
increased rainfall and the number of monthly cases
Exposure to harmful UV irradiation (240 - 380 μW/cm2 UV; <10% of bacterial cells
are viable after 10sec)
Centre Testing International (CTI)

43. ENVIRONMENTAL SURVIVAL
Legionella cells live intracellularly as
protozoan parasite and nematodes:
- Increase environmental survival and water
disinfection resistance
- (protozoa) Vehicle of transmission
Cells are able to grow and participate
in multispecies biofilm
The presence of sediment, sludge, scale,
rust also play an role in harbouring and
providing favourable conditions in which
Legionella may grow
Hilbi, H et al. Mol. Microbiol. 2010
Animal model

44. EPIDEMIOLOGY
Legionnaires’ disease is a relative uncommon, mainly sporadic respiratory infection
with low notification rates in EU/EEA countries
- overall 1.4 per 100 000 inhabitants in 2014
- True incidence estimated 20x; many community acquired infection undreported
Five countries (France, Germany, Italy, Portugal and Spain) accounted for 74% of
notified cases
In 2014 one of the largest outbreak involving more than 400 cases occurred in Vila
Franca de Xira near Lisbon, Portugal, have been reported
Regular checks for Legionella and appropriate control measures in man-made water
systems may prevent a significant proportion of Legionnaires’ disease cases.

45. EPIDEMIOLOGY
Seasonality – summer/fall
The peak months in Europe for the onset of
Legionnaires’ disease occur during the summer,
the period when most people take their main
holiday
Most elderly age group experiencing the
highest incidence of Legionnaires’ disease
Accommodation sites during holidays
Cases associated with travel are known to comprise up to 50% of national
reports of the disease
Sex distribution

46. RISK-FACTORS
Man over 40 with underline health issues à the most susceptible
population for community acquired and travel-associated infections
-Smoking
-Alcohol abuse
-Diabetes
-Heart diseases
-Reduced immune competence
Nosocomial legionnaires’ disease
-Reduced immune competence, cancer, lung diseases, treatment with
respiratory devises

47. OUTBREAKS
The two largest registered Legionella community-associated outbreaks
(Mursia, Spain, 2001 – 800 cases; Portugal, 2014 – 400 cases) were related
to cooling towers
DW-borne outbreaks usually occurred in large buildings or institutional
settings and were related to multiplication of Legionella in the respective
distribution system
DW-borne (not outbreaks) of legionellosis à community acquired or sporadic
depending on the risk status

48. THE INFECTIVE DOSE PARADOX
Limited data regarding human dose response for L. pneumophila in human
The concentration of Legionella required to result in an outbreak is unknown
Organism is ubiquitous to many natural and artificial environments which
suggest people are frequently exposed to low concentration
Low concentrations of legionellae seem to be emitted from water systems,
and epidemiological evidence indicates that infection can occur at some
distance from the source of aerosol
Legionella spp. represent a health risk to humans when a concentration of
104 to 105 CFU per liter potable water is exceeded
Whiley et al. Front Microbiol. 2014; Delgado-Viscogliosi et al. 2005; O’Brien and Bhopal. Lancet 1993

49. CONTROL IN WATER SYSTEM
Difficult to achieve sustainable long-term elimination from aquatic environments
- Different cases of Legionella re-emergence after (hyper-)chlorination
- Amoebae are suspected to be involved in the resuscitation of Legionella that have entered a VBNC
state after disinfection
Maintaining the water temperatures above at least 50°C in hot water systems with
occasionally increases to 65°C might be considered an effective control measure
- Increase heat tolerance of Legionella inside thermotolerant free-living amoebae
- Biofilm increase heat tolerance
Removal or reduction of biofilms and protozoa is essential when trying to
reduce Legionella levels