https://pubmed.ncbi.nlm.nih.

gov/11541193/#:~:text=The%20amount%20of%20biocide%20needed,of
%20cellular%20material%20is%20required.

Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of
spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal
and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the
water distribution system of the space shuttle Discovery, was grown in continuous culture to
produce a bacterial contamination source for biofilm formation and removal studies. A 10(7)
CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000
micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis
and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and
9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with
oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image
analysis. Fifty percent of the biofilm material was removed in the first hour of continous
treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any
measurable cellular material after 6 h continuous contact. After this first removal of biofilms by
the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine
was the only compound that was unable to remove cellular debris from either primary or
secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of
formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested
inactivated the B. cepacia associated with both primary and secondary biofilms. The amount of
biocide needed to inactivate 50% of planktonic B. cepacia in 10 min at 25 degrees C was 8.4, 0.5,
and 0.2 mg l-1 for iodine, chlorine, and ozone, respectively. The data suggest that iodine maynot
be the best chemical for treating of biofilms when removal of cellular material is required.

https://www.sciencedirect.com/science/article/abs/pii/S0195670108003551

Comparing the chlorine disinfection of

detached biofilm clusters with those of sessile
biofilms and planktonic cells in single- and
dual-species cultures

Risk Mitigation Steps to Exclude B. cepacia from

Non-Sterile Drug Products
Risk mitigation steps to exclude B. cepacia complex include:

 Pharmaceutical ingredients selection

 Product formulation including robust antimicrobial preservative system
 Management of pharmaceutical water systems
 Equipment cleaning and sanitization
 Manufacturing processes
 Risk-based microbial testing programs

When selecting pharmaceutical ingredients the microbiological quality of these ingredients may be

important depending on the dosage form. Keep in mind the quantity of the ingredient used in the

product, the manufacturing process of the ingredient, physical attributes of the product especially

water activity, the antimicrobial effectiveness of the formulation, and the intended use of the

product. There is a hierarchy of risk for microbial contamination with chemically synthesized

ingredients having the lowest risk and animal-derived ingredients the highest risk. The hierarchy is

animal-derived > plantderived > mineral-derived > semi-synthetic > synthetic ingredients. This

generalized sequence can be modified by the extent of processing during manufacturing (Cundell,

2005).

Multiple-use non-sterile products must be resistant to microbial contamination and growth due to

their low water activity, i.e., <0.6, inherent antimicrobial activity or to the effectiveness of their

antimicrobial preservative system. During product development, candidate formulations are

assessed using the methods described in USP <51> Antimicrobial Effectiveness Testing. As

recommended in the USP chapter, additional challenge organisms, including members of the BCC,

can be added to the test to ensure the formulation has the most robust preservative system.

Investigations of BCC infection outbreaks have consistently identified the pharmaceutical-grade

water system at the manufacturing site to be the probable cause of the product contamination.

Manufacturers of aqueous non-sterile drug products should install a well-designed water system,

and well maintain and manage the system aggressively.

Process equipment including tanks, pumps, and filling lines, especially their product contact surface

must be adequately cleaned and stored dry to avoid product contamination. Microbial surface
monitoring must be included within standard equipment cleaning protocols and periodic cleaning

verification implemented.

For each dosage form, the unit manufacturing operational steps can be analyzed for potential

microbial contamination risk. For example, for an oral liquid the manufacturing steps are ingredient

procurement, equipment cleaning, weighing, blending, mixing, container-closure preparation, and

filling. Ingredient water and processing equipment cleaning may be viewed as areas of greatest risk

for microbial contamination. Another factor may be the order of addition of the preservative, as they

must be present in the aqueous phase of an oil-water suspension. For a comprehensive discussion

of microbial contamination of non-sterile drug products, the reader is referred to the USP General

Informational Chapter <1115> Bioburden Control of Non-sterile Drug Substances and Products.

When USP <60> becomes official, whether a microbiological specification for the absence of B.

cepacia complex will be added to any drug product monographs will remain to be seen. A risk-based

microbial testing program would involve more frequent testing of aqueous drug products than non-

aqueous products. For example, based on a comprehensive risk assessment and a testing history, a

compressed tablet may not have a microbiological specification and would not be subjected to

release testing, whereas each batch of a topical cream would be subject to full testing as

recommended in USP <1111>.

Conclusions
Although microbial contamination of drug products results in few patient infections, the

pharmaceutical industry must not be complacent working to eliminate this risk. The author hopes

that this review article contributes to this goal through a timely discussion of the role that BCC plays

in microbial contamination and how it can be detected.

B. cepacia complex species give off a distinctive odor described as dirt-like in the Manual of
Clinical Microbiology. The species can form biofilms by using mucin-binding proteins. The
organism is resistant to many common antibiotics.

https://www.ijmronline.org/html-article/11053
https://www.ijmronline.org/journal-article-file/11053

a)- Bacterial samples obtained from tanks and machine filling.

Before each prepared batch we sampled the final rinse from 10 points (syrup tank –
hold tank – buffer tank – pipe – 6 nozzles) so we have 210 samples depending on
number of prepared batches in Stage I (21 F).
190 samples out of 210 were contaminated and identified as the same contaminant
organism.
"Burkholderia cepacia" as in the aqueous finished products in Stage I although the
washing and sanitization of these tanks and nozzles by ethyl alcohol 70%.
After these important findings, we conclude that the source of contamination by a
percentage 90% is the machine preparation and hold tanks and the machine filling
pipes and nozzles.
We should disinfect all of these sites by a new disinfectant which has a strong
bactericidal effect especially on Burkholderia cepacia.
b)-Disinfectant efficacy test
Tego (Alkyl diazapetane) is used for the disinfection of floors, walls, work surfaces and
process equipment and it is applied to the surface.
As the log reduction is more than 3 as the disinfectant Tego 2000 has a great
bactericidal and fungicidal effect against organisms at 15 minutes on all surfaces, so it
is recommended for using this disinfectant instead of ethyl alcohol 70% (Table 2).
Viragri plus is based on an optimized blend of glutaraldehyde and quaternary
ammonium compound (QAC) in aqueous solution. It is a highly effective, non-oxidizing
disinfectant specially formulated for use on cleaned surfaces in the sectors where
biocidal activity is required like pharmaceutical industry applications.
As the log reduction is more than 3 as the disinfectant Viragri plus has a great
bactericidal and fungicidal effect against organisms at 15 minutes on all surfaces (Table
3).
At the end of this stage we conclude that the rinsing with these two disinfectants on all
the machine tanks and filling parts with planned rotation instead of ethyl alcohol 70%
only will be the efficient solution to prevent the recurrence of all the contamination by
Burkholderia cepacia.
Accordingly monitoring and examining the produced batches after this treatment is very
important as shown in the third stage.