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Microbial Contamination in Water Systems

Fritz Roeder and Tim Sandle

PDA Journal of Pharmaceutical Science and Technology 2022,

Access the most recent version at doi:10.5731/pdajpst.2021.012636
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Title Page

Manuscript Title:
Microbial Contamination in Water Systems

Authors:

Fritz Röder (Corresponding Author)

Merck Healthcare KGaA
Frankfurter Str. 250
64293 Darmstadt
Germany
[email protected]
06151/72-57929

Dr. Tim Sandle

Bio Products Laboratory Limited
Dagger Lane
Elstree Herts
WD6 3BX
U.K.
02082582483
[email protected]

Key words:

- Water
- Sanitization
- GMP
- Biofilm
- Microbiology
- Purified Water
- Water for Injection

Citations see last page

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Microbial Contamination in Water Systems

Abstract:
The microbiological impurities are the most critical ones in a water system and are thus of
special concern. In case of contamination of a purification unit or a distribution system, the
user must react quickly. The authors have written down their experience and explain details
about the typical microbiome of a water system. In addition, advice is given on how to
remediate microbial contamination.

Water is one of the most important bacterial habitats on Earth, showing considerable
biodiversity, and water is a major means for the dissemination of microorganisms(1).
Globally, in fresh water, the most predominant bacteria belong to the
phyla Proteobacteria (mainly of the classes Alpha-, Beta- and Gammaproteobacteria),
Actinobacteria, Bacteroidetes and Firmicutes, irrespective of the type of water surface (lakes,
rivers, wetlands), mineral, drinking or wastewater. While some organisms cannot be
cultivated (2), traditional microbial methods can detect sufficient numbers of bacteria to
indicate the status of a water system with respect to contamination control..

The water treatment process by municipal water authorities will ideally be effective at
reducing down a level of microorganisms and in many parts of the world, water suppliers
have statutory duties to ensure that there are no faecal indicator bacteria present. However,
controls in some parts of the world are less stringent and it may be prudent to perform testing
for indicator organisms such as Escherichia coli. Even where good controls of the supplied
water are in place, the inlet water supplying the pharmaceutical facility will be controlled in
many parts of the world to 500 CFU/mL (such as in the U.S. and Europe). However, in other
territories supply water may contain several thousand microorganisms per millilitre (despite
stricter governmental requirements) and the purification process must be appropriately
designed to remove these organisms and, where applicable in the case of Water-for-
Injections, endotoxic by-products. Therefore, the design, operation and maintenance of
pharmaceutical-grade water systems is critical for ensuring suitable in-process control and
final product quality. The levels of microorganisms in the in-coming water can be hard to
predict, due to the variables of seasonality particularly for countries with temperate climates),
temperature, pH, velocity, stresses and heat, plus differences in water system design between
one facility and another.

Microbial ecology of pharmaceutical water systems

The basics of biofilm generation and detachment are well-described in PDA TR 69(3). In
terms of the most common microorganisms found associated with a pharmaceutical water
system, Sandle undertook a meta-study as a longitudinal analysis of the organisms typically
recovered from mains water, purified water and WFI (4). For this, samples of water were
assessed over a ten year period using Plate Count Agar (incubated at 30-35oC for five days)
and R2A agar (incubated at 20-25oC for seven days). These culture media and incubation
parameters had been previously assessed as the most likely to cultivate the highest numbers
and greater diversity of organisms. The primary genera from purified water systems were
found to be (top 4 only):

• Ralstonia 30%
• Burkholderia 23%
• Pseudomonas 14%
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• Moraxella 7%

The primary species were identified as (in order of occurrence): Ralstonia pickettii,
Burkholderia cepacia, Moraxella spp., Stenotrophomonas maltophilia, Ochrobactrum
anthropi, and Pseudomonas oryzihabitans.

With WFI, recoveries were lower. The primary genera were identified as (top 4 only):

• Pseudomonas 13%
• Burkholderia 6%
• Ralstonia 6%
• Moraxella 4%

The main species in WFI were (in order of occurrence): Ralstonia pickettii, Burkholderia
cepacian, Pseudomonas oryzihabitans, Moraxella spp., and Pseudomonas fluorescens.

The genera recovered between purified and WFI systems showed similarity, and all
recovered organisms are morphologically Gram-negative rods. In both cases, due to the
limitations of culturability the data will be an underestimation in terms of numbers and there
may be some species present that are not recoverable on standard growth media, like R2A(5).

The most important question stemming from this relates to which of these common
organisms are most resistant to different method designed to sanitize water systems, which is
the central question that this paper addresses. Table 1 of this article shows how to remediate
some of these species when found in water systems.

An additional challenge arises from the majority of bacteria present in pharmaceutical water
systems existing as biofilms, adhering to equipment surfaces. With biofilms, bacteria are
attached to surfaces and protected within a slime-like matrix (the “Extracellular Polymeric
Substances matrix”). Bacteria within these types of communities are harder to kill than those
in the free-floating (planktonic) state due to proteinaceous barriers existing to chemical
penetration and heat inactivation(6). All that is required for biofilm formation is a given
concentration of planktonic bacteria, plus water, plus a surface, plus available nutrients
(including a carbon source) or a contaminating event that led to a biofilm development,
leading to the gradual release of organisms(7). A purified water system will have the required
level of nutrients for a biofilm to develop; the risk is where there is a pipe dead-leg, pipe
elbows where there is less than 90 degree angle, or some other factor that causes the water
level to slowdown, causing the flow to move from turbulent to laminar. In practice, detailed
engineering know-how is required to design a distribution loop with a perfect balance of
sufficient turbulence and low energy consumption of the pumps in case of high water
withdrawal.

To guard against biofilm formation, many systems have controls in place to prevent biofilm
development including sanitization using ozone, hot water, or chemicals. An additional factor
is with the material from which distribution pipes are constructed. For example, cement-
based materials (cement and asbestos) support fewer fixed bacteria than plastic-based
materials (such as PVC, uPVC and MDPE) (8). Metal, in the form of highly-polished
stainless steel, also reduces the likelihood of bacterial attachment, although this is property
diminishes should corrosion or rouging occur(9, for example due to lack of passivation.
However, should a biofilm develop then an out-of-control situation may emerge. Where a
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biofilm develops in the distribution loop then the formation can often only be detected from
point-of-use samples, or from sampling ports along the distribution loop. Several excursions
are required to alert of the probability of a biofilm since the release of bacteria from a biofilm
is variable (together with variabilities of sampling). By the time a biofilm is suspected,
through microbial recovery from sampling, the biofilm will most likely have become
significantly established. In addition to point-of-use microbial sampling, biofilms are not
straightforward to detect, although there are methods available such as colorimetric staining
(where chemicals are used selectively stain the protective matrix of biofilms) or extraction of
surface materials for study using transmission electron microscopy. Such methods are often
limited by much of the distribution system being inaccessible. Additional insights could be
gained from rapid microbiological methods, such as flow cytometry (10) and vibrational
spectroscopy via infrared or Raman spectra (11), to enable real-time assessments. These have
not been deployed by the authors; however, it is anticipated that further technological
advancements will provide better insights into biofilm presence or the rate of organism
shedding from biofilm communities.

Anti-microbial water treatment

Anti-microbial water treatments can be divided into the physical (heat) and chemical (such as
the application of ozone, chlorine, chlorine dioxide, hydrogen peroxide, peracetic acid and
sodium hydroxide). There are different advantages and disadvantages when applying these
treatments, and there are different levels of bacterial resistance (both innate to the organism
and in relation to the organisms presence in a biofilm where protection is conferred by
exopolysaccharides). The assessment of the resistance of microorganisms in water is
important since some microorganisms become more resistant in low nutrient environments,
compared when they are in media with a rich supply of nutrients(12).

Ozone

Ozone is common method of water disinfection, especially generated and circulating

distributed purified water. Ozone is a bluish gas and at atmospheric pressure, ozone can
partially dissolve in water, where it is relatively unstable with a half-life of 15 up to 60
minutes Because of its relatively short half-life, ozone is always generated on-site by an
ozone generator. Care needs to be taken with the management of ozone systems as not to
corrode gaskets or seals and any residual ozone must be removed (such as by applying a 254
nm UV light) before the water is used for pharmaceutical processing.

The importance of processes like ozone for the regular disinfection of water is because filters,
if they are used, are not wholly effective for maintaining microbial control (the use of filters
cannot be the only microbial control mechanism due to the risk of the water becoming re-
contaminated). With the use of filters, many regulatory agencies advise against their use and
require a stated purpose, supporting validation data and an SOP with the change out
frequency assessed. Moreover, microorganisms are readily able to colonize the surface of a
filter or reverse osmosis membrane where growth can accelerate due organisms utilising
assimilable organic carbon concentrations.

The effectiveness of ozone relates to its high oxidation potential (2,07 eV) where the process
can oxidize cell components of the bacterial cell wall, provided that the contact time is
sufficient for cell wall penetration to take place. On entering the cell, ozone will oxidize all
essential components (such as enzymes, proteins, DNA and RNA). Furthermore, where the
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cellular membrane is damaged (lysis), much of the cell material will be removed and the cell
will fall apart. Ozone is theoretically effective in killing all types of bacteria, provided that
the concentration is sufficiently high and optimized for a given water system. However, if a
biofilm has formed in a water system, the mentioned EPS matrix starts to build up and protect
microorganisms. The microorganisms below the matrix are affected less by the ozone. In
other words, ozone can be effective in preventing biofilms but not in their removal. So, if
contamination has already taken place, a sanitization procedure using sodium hydroxide or
heat may be the better choice.

The most commonly used model to describe water disinfection by ozone is the Chick-Watson
law, which is the disinfectant concentration multiplied by the contact time. The same
approach is used for assessing chlorine levels. This law can be mathematically represented as
follows:

k = Cn * t
Where:

• k = reaction-constant, dependent on the type of microorganism and the disinfectant

• C = disinfectant concentration
• t = contact time, period of time that the disinfectant is in contact with water
• n = constant

In most cases n equals 1, causing the deactivation of bacteria to become a first-order reaction.
This formula can be used to determine which microorganisms are most resistant to ozone
treatment.

Microorganisms resistant to ozone

No mechanism has been established where microorganisms become resistant to ozone.

However, the minimum inhibitory concentration required to exert an antimicrobial effect
varies across different species and the antimicrobial effect can also be inhibited by the
presence of impurities in the water (13). Hence, ensuring appropriate concentration and
contact time is important. During disinfection, the Ct-value can be applied (as discussed
above). This value is a multiplication of the disinfectant concentration (C) in mg/L and
contact time (t) in minutes, required to deactivate a population of microorganisms. With
ozonation, Gram-negative bacteria have been shown to be more resistant than Gram-positive
bacteria. For example, Pseudomonas aeruginosa requires twice the contact time as
Staphylococcus aureus in water to achieve the same log reduction(14). Furthermore, C-t
values for ozone can be affected by different factors, such as concentration and time; together
with the pH value, sunlight, water temperature, mixture of water and the disinfectant, and
contact chamber design. Provided that the ozone concentration and contact time is sufficient,
and all variables are controlled, ozonation will be sufficient to achieve around a theoretical 6-
log reduction of planktonic microorganisms in water. In addition, it is crucial to remove
remaining concentrations of ozone or any other chemical agents after sanitization as much as
possible. Any remaining agents in the water might have an influence on the active substances
used in manufacturing.

Chlorine
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Chlorine penetrates bacterial cells by diffusion. Once within the cell, chlorine will affect
different enzyme types. Effective chlorination can be achieved with minimum levels of 0.2
mg/litre of free chlorine(15). Just below this level, experimental data has shown that more
resistant microorganisms can survive a 2-minute exposure to 1.0 mg of free chlorine per litre.

In terms of application, there are some concerns with the use of chlorine due to compatibility
issues with higher quality stainless steel (such as 316L grade). Due to compatibility issues, it
is less common for chlorine to be used to decontaminate WFI or purified water systems, and
hence for chorine to be more commonly applied to potable water or to the early stages of
water treatment processes.

Microorganisms resistant to chlorine

As with ozone, different species of bacteria have different levels of resistance to chlorine in
terms of contact time and concentration. Due to this, maintaining chlorine levels is important
since selection for more chlorine-tolerant microorganisms in chlorinated waters. As an
example, Mycobacteria are highly resistant to chlorine and the other chemical disinfectants,
and exposure times of up to 8 hours are required(16). In general, Gram-positive bacteria are
more resistant than Gram-negatives, a fact that offers some control assurance as these types
of organisms are less common to pharmaceutical water systems. Furthermore, Aeromonas
hydrophila can be resistant to standard chlorine treatments, especially within a biofilm
community(17). Other Gram-negative organisms with slightly higher resistance are species
Klebsiella, Pseudomonas, Alcaligenes, Flavobacterium, Moraxella, and Acinetobacter.
However, each of these can be killed by 10 mg of free chlorine per litre, provided the contact
time is sufficient(18). Some bacteria are additionally developing resistance to chlorine in a
manner that parallels antibiotic resistance through gene overexpression leading to efflux
pumps in Pseudomonas species being able to export chlorine from the cell (19). Therefore,
complete removal of chemicals after sanitization is essential.

Chloramine

Chloramines are derivatives of ammonia and are used to treat some public water supplies as
an alternative chlorine (typically as monochloramine) because these chemicals are less
oxidative and less reactive than chlorine, it is less reacted and therefore more stable it does
not dissipate as rapidly as free chlorine. The use of chloramines is not common for use with
pharmaceutical water systems.

Microorganisms resistant to chloramine

Bacterial populations in drinking water have been shown to exhibit differential resistance to
monochloramine, and it is apparent that the disinfection process selects for resistant bacterial
populations. This is through some bacteria surviving treatment and through the transfer of
genes (20), similar to exhibited resistance to chlorine. Organisms showing greatest resistance
Pseudomonad-types such as Sphingomonas species(21).

UV light

Water can be subject to ultraviolet light (UV-C) to inactivate microorganisms, especially for
circuiting purified water. Effectiveness is based on exposure time (typically, microwatt-
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seconds per square centimetre) and lamp intensity. Most water-associated microorganisms
are destroyed under different wave lengths and intensities, typically irradiation at 254 nm,
high intensity (lower intensity UV is needed to remove ozone).

1. Chlorine removal - irradiation at 254 nm, very high

intensity;
2. Disinfection - irradiation at 254 nm, low intensity;
3. Reduction of total organic carbon - irradiation at 185 nm and 254 nm,
medium intensity;
4. Removal of ozone - irradiation at 254 nm, medium intensity.

Inactivation occurs by UV light destroying nucleic acids and disrupting microbial DNA(22).

Microorganisms resistant to ultraviolet light

Most Gram-negative bacteria are susceptible to UV light through destruction of nucleic acids
and disruption of DNA (23), provided the light is at the correct wavelength and the exposure
time is sufficient. Within the Gram-negatives, Pseudomonads like Pseudomonas aeruginosa
exhibit greater resistance than coliforms. Most microorganisms common to water systems can
be inactivated. Issues occur when UV lamps lose power or where they are not properly
maintained, or if the flow rate increases so that the required contact time is not achieved.

Heat

Heat is an effective means to control bacteria in water and where the water temperature is
maintained above 65oC (many systems are maintained between 70 and 80°C) this will be
sufficient to kill all microorganisms present in a typical water system (thermophilic
microorganisms will not be present, based on the typical unsuitability of the pharmaceutical
environment to such organisms). The application of heat to water distribution loops means
that the water is subject to continuous sanitisation. Where a biofilm develops, hot water is
generally effective. Under rare circumstances, to overcome any heat resistance within the
biofilm community super-heat can be used. This is undertaken by elevating the temperature
within the affected region to 121°C and ensuring the temperature is held for a sufficiently
long contact time (typically 10-15 minutes) so that the heat an penetrate any biofilm matrix
present(24). Heat is also an effective method, compared with chemicals, for the treatment of
biofilms. The risks associated with water temperature arise either where water temperature is
not maintained in the circulating loop or when water is cooled so that personnel can use the
water (such as through heat exchangers). With heat exchangers, inadequate heat time for
sanitising a cooling heat exchanger thoroughly can lead to operational problems. Further with
the cooling of water, provided the heat exchanger is well maintained and not subject to
leakage, the primary risk is at the outlet point where poor tubing management or inadequate
flushing can lead to contamination build-up.

Microorganisms resistant to heat

While different species of bacteria have their own heat tolerance, as indicated above, no
typical microorganisms found in pharmaceutical water systems can survive temperatures of
70oC or above. The risks arise when temperature is not maintained, where lower temperatures
are used, or with cold water systems. In relation to heat, cold water systems will allow most
water-associated microorganisms to survive and thrive(25).
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Protective mechanisms

With chemical treatments, microbial resistance is greater for organisms attached to surfaces
compared with those in the free-floating state(26). With biofilm communities, by being
formed of different species naturally occurring biofilms are more resistant to disinfection than
a single-species biofilm (as might be created in a laboratory setting)(27); and older biofilms
contain more resistant communities than newly formed ones(28). Biofilms are notoriously
difficult to remove; what is of great importance is designing and implementing effective
control strategies to prevent biofilm formation taking place. This includes the importance of
ensuring that regular disinfection takes place. For example, a depletion of chlorine in supply
water being processed in a water generation plant can lead to a four-fold increase in biofilm
formation. Further factors that can promote resistance are organisms growing in nutrively
depleted states and those which can undergo encapsulation (forming a polysaccharide layer
that lies outside the cell envelope).

Due to different resistant patterns afforded by bacterial genera, surfaces, age of a biofilm,
encapsulation, and nutrient effects, plus different mechanisms of action for different
disinfectants, a multiple strategy of disinfection using heat and chemicals, or by using more
than one chemical, maybe required in cases of water system contamination. This is also
fostered by the new EMA Q&A document on water for injection. (29)

Implementation of a sanitisation concept for a water system

All the above discussed aspects must be considered during the design phase of a water
system. Usually, the discussion starts with the distribution temperature in the loop. One main
factor that influences that important question is the needed amount of hot and cold water at
the points of use (POU’s). If many use points need hot pure water, a hot distribution loop
may be chosen. The disadvantage is the high cost for heating, but this is considered as a very
safe method for preventing biofilm formation. It may also be possible to only heat up the
water periodically to save energy costs. A second possibility is the use of ozone in the loop.
Here, high invest costs occur, including expensive spare parts (ozone generator, measuring
technology). But this approach has also been proven in practice to be very effective.

Concerning sanitization of the water generation system itself, ozone cannot be used. An
exception is ceramic ultrafiltration modules which have been recently applied the first time
for membrane-based WFI(30). But for the rest of the entire purification unit, a different
approach must be used. The two options for sanitizing the purification unit are periodic hot
water sanitization or dosing of chemical substances. The current technology can withstand
temperatures up to 80°C, some unit operations also higher. As chemical substances, hydrogen
peroxide, peracetic acid or several chlorine compounds may be used (chlorine not for RO
membranes). The practice has shown over decades that hot temperatures provide the best
results against a broad variety of species. Moreover, temperature-controlled sanitization is
very easy and cost-effective to automate and to be carried out in times of no production in the
facility.

When a water treatment system is designed, the microbiology must be considered as one of
the most critical quality attributes of the produced water. Therefore, the user must choose a
suitable level of assurance to control bacteria und find a suitable design. Critical aspects
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concerning the control strategy are the source of the feed water and also the desired product
water quality. Unfortunately, for smaller water systems (such as in laboratories), better
solutions for biofilm control can often not be afforded. Consequently, smaller systems are
commonly more difficult to operate and to be kept in a state of control (same water is
recirculated several times, less dilution, less measurement technology available).

Reliable operation of water systems

Once a system has been constructed, installed and passivated at the desired location, it is
filled with water the first time. This is the moment from when the user must keep on
operating the water system and controlling it. The only possibility to stop it again and to
prevent microbial proliferation is to completely empty and dry the entire system. In case of
the use of softeners, the resins can never be entirely dried again without replacing them. So,
the only possibility here would be to withdraw the resin from the pressure vessel. The same
problem occurs when using activated carbon.

During system qualification, a suitable sanitization frequency of all parts is developed. The
needed concentration of disinfectant agent / respective temperature as well as the frequency
and duration of the entire procedure are subject to the performance qualification. Any
frequency must be proven to be effective. When adjusting sanitization parameters later during
normal operation, a change qualification is needed. Typical sanitization frequencies vary
between weekly and 8 weeks. In case of manual procedures for smaller systems (e.g. periodic
dosing of peroxide) a longer frequency is often used due to a high workload and the manual
handling of chemicals. But this also goes along with an increased contamination risk.

In case of microbial proliferation inside the system

With nearly any pharmaceutical company, users can tell of their experiences they have with
increased microbiological results in their water systems. But a really systematic approach
with how to remediate a biofilm is normally not available, often due to users lacking
experience in this area. Therefore, such considerations are described adjacently.

According USP 1231(31), a biofilm would at least take two weeks to develop. In case of
increased results in any use point (or in the purification unit), the first step should be a
resampling of the concerned POU to preclude any sampling failures. In parallel, the
identification of the genus or species of each isolated contaminant needs to be started. If the
routine sampling frequency at this point of use is longer than two weeks, then an increased
for-cause sampling procedure should be conducted.

Once it is confirmed that microbial proliferation is in progress, time becomes critical. The
longer the user waits to remediate the problem, the more difficult it gets. Depending on the
species, different sanitization practices may be applied. One way that has brought good
results in practice to combat a broad range of microbial species is the “3-13-30” rule: increase
pH for 3 hours to a value of 13, with increased temperature to 30°C. Of course, it must be
checked in advance if all parts of the system are capable to withstand these conditions.
Typically, caustic soda is used to leverage the pH (as an alternative to sodium hydroxide). If
no heat exchanger is installed in the system, the same procedure may be carried out at
ambient temperature.
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Nevertheless, some bacteria are less heat resistant. Depending on the found species, hot water
sanitization can also provide very good results. In addition, heat distribution throughout the
entire system is much easier to achieve than distribution of a sanitizing agent. However, it is
admitted that the heat kills the bacteria but does not oxidize them. Consequently, the
remaining dead bacteria still provide a source of nutrient and can support the build-up of a
new biofilm. Therefore, a combined approach using first heat and then chemical agents may
help killing and oxidizing the biofilm as well. In total, the right sanitization method depends
on the type of contamination and on the species found.

The last option is to replace parts of the system (or the entire system) that cannot be sanitized
with any above-mentioned approaches. The following table (Table 1) provides some
experiences of the authors with different species and how to remove these types of
bacteria.(32)

[Insert Table 1 here, see last page]

Summary

This paper has considered the main microbial contaminants within pharmaceutical water
systems, based on a longitudinal study and has considered how systems can become
contaminated (principally a combination of poor design or poor maintenance). The focus of
the paper then shifted to considered remediation methods for addressing microbial
contamination of mater systems, dividing such methods into chemical, physical and heat.
Special attention was paid to biofilms and the particular problems that biofilm contamination
presents. In evaluating the different remediation methods, it was noted that some types of
methods (and variation within the methods such as differences in temperature) are required to
be enacted against different species of organism. Overall, heat remains the most effective
method and it is good practice to either use hot water systems or, where cold water systems
are used, to have in place a mechanism to heat the water periodically or where microbial
contamination occurs or is suspected. In case of remediation of existing biofilms, a
combination of heat and NaOH shows good results.

The Authors hereby declare having no financial or nonfinancial competing interests related to
the manuscript.

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Tables:

Table 1: Different microbiological species and their remediation in a water system; the
highlighted ones were also identified in the study mentioned above (3).

Genus / Species Gram Habitat Comments Remediation

stain
Pseudomonads Negative Water, soil, So-called Heat or chemical;
environment “superbug”; human check species, this is
(ubiquitous) pathogen; this is a a genus.
genus
Pseudomonas Negative Water, soil, Can proliferate at 9- Heat: minimum 60
aeruginosa environment 42°C; Inactivation at min at 55°C;
(ubiquitous) 55°C; temperature chemical methods
optimum at 37°C may also help.
Enterobac- Negative Gastrointestinal Facultatively Heat or chemical;
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Genus / Species Gram Habitat Comments Remediation

stain
teriaceae passage, waste anaerobic metabolism, check species, this is
water, soil often coliform; this is a genus.
(ubiquitous) a genus. Common
species are Klebsiella,
Salmonella, E. coli
Ralstonia Negative Ubiquitous Good proliferation at Very resistant
30°C, bad against chemicals.
picketii
proliferation at 37°C When this is the
and higher; can case, try heat.
survive in disinfectant Consider replacing
containers; difficult system parts.
to incubate
Eschericha coli Negative Gastrointestinal Coliform bacteria; Resistant against
passage, food, faecal organism; chlorine, but not
water relevant in potable against ozone or heat
water; used as (> 60°C helps)
indicator species; ideal
proliferation at 37-
42°C
Legionella Negative Ubiquitous in Rod-shaped; genus; Use heat above
water for potable water, 60°C..
Legionella
pneumophila is most
relevant; ideal
proliferation at 35-
45°C; good
proliferation in
nebulized water
Enterococcus Positive Gastrointestinal Indicator organism in Species as high
spp. passage, food, potable water tolerance for salt and
environment chemicals; try heat.
Acinetobacter Negative Ubiquitous in “Superbug”; good Use Heat (80°C).
baumanii water proliferation at pH 5-6
and in warm water
Pseudomonas Negative Water, soil, Good proliferation at Heat: minimum 60
oryzihabitans environment 25-30°C, bad min at 55°C;
(ubiquitous) proliferation at 37°C chemical methods
and higher. such as ozone are
also effective
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