eBook: TOC and Microbial Detection Monitoring - 33

Biofilm, Endotoxins and Their Relationship

not penetrate into the base layers of the biofilm but only
interacts with the top layer.

ETTLER TOLEDO White

If a water loop is operated at a high flow rate, it will
reduce the shedding of the flocs or planktonic bacteria.
However, while the high flow rate may reduce shedding, it also exposes the biofilm to more nutrients and
could result in an increased growth rate for surface
hugging biofilm.
A biofilm layer is a complex aggregation of microorganisms marked by the secretion of extracellular
polymeric substances (EPS) that act as an anchor
to the surface as well as to other microorganisms
of various species. Biofilm communities actually
communicate, using a process referred to as quorum
sensing, and in the presence of danger or death
(the "flight or fight response"), secrete protective
metabolites within the three-dimensional community structure that creates a form of protection to the
layers within the biofilm. Thus, if the top layer of a
biofilm community is exposed to Hot WFI, the lower
layers can be protected by the metabolite layer and
will continue to multiply.

Figure 2: A pressure sensor with biofilm build up.

biofilm was sheared off and became planktonic in the
water system.
This example of a Hot WFI system that was not exhibiting colony forming units when tested with the traditional method, provides the connection to the relationship
between standard plate counting and LAL testing.
Endotoxins and biofilm
Endotoxins are a type of pyrogen or fever-causing agent
and are a lipopolysaccharide (LPS) component of the
exterior cell wall of Gram-negative bacteria, such as
E. coli. Endotoxins are part of the outer membrane of
such bacteria whether the organisms are pathogenic
or not. Although the term "endotoxin" is occasionally
used to refer to any cell-associated bacterial toxin, in
bacteriology it is properly reserved to refer to the LPS
complex associated with the outer membrane of Gramnegative pathogens such as Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus
influenzae, Bordetella pertussis and Vibrio cholerae.

Organisms in a Hot WFI system may be in a stressed
state, be spore formers, or Viable but Not Culturable
(VBNC) with standard methods and it can be very
difficult to capture enough viable organisms to culture
with standard plate count methods. However, viable
organisms, VBNCs and spores will be detected by an
on-line microbial detection analyzer.
Shown in Figure 2 is a pressure sensor that was removed from a pharmaceutical company's Hot WFI system
for inspection of the gasket. Examination of the part
clearly revealed it was covered with biofilm that was
surviving and growing. However, the standard grab
sample method that was being used by the company
measured 0 or 1 CFU with most incubated plates. In
this situation, an on-line microbial detection analyzer
would have detected bioburden from the sensor as

The current pharmaceutical test for endotoxins is the
LAL test and is required for WFI and Pure Steam. LAL
reacts with bacterial endotoxin lipopolysaccharide
(LPS), which is a membrane component of Gramnegative bacteria, as previously discussed.

33
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