ASTM E3321-21

Standard Test Method for Intraluminal Catheter Model used to Evaluate Antimicrobial Urinary Catheters for Prevention of <emph type="ital">Escherichia coli</emph> Biofilm Growth

SIGNIFICANCE AND USE
5.1 In the battle to reduce medical device and implant-related infections, prevention of bacterial colonization of surfaces is a logical strategy. Bacterial colonization of a surface is a precursor to biofilm formation. Biofilm is the etiological agent of many implant and device-related infections and once established, microorganisms in biofilm can be up to 1000 times more tolerant to antibiotic therapy. Often the best treatment strategy is removal of the implant or device at a high socioeconomic cost. Catheter associated urinary tract infections (CAUTI) are the most prevalent of the device-related healthcare associated infections. Catheter associated infections account for 37 % of all hospital acquired infections (HAI) and 70 % of all nosocomial urinary tract infections (UTI) in the U.S. (2, 3). The Intraluminal Catheter Model (ICM) was developed to evaluate the ability of antimicrobial catheters to inhibit biofilm growth on the catheter lumen.  
5.2 The purpose of this test method is to direct a user in how to grow, sample, and analyze an E. coli biofilm in a urinary catheter under a constant flow of artificial urine. The test method incorporates operational parameters utilized in similar published methods (4). The E. coli biofilm that grows has a patchy appearance that varies across the catheter. Microscopically, the biofilm is heterogenous, with large clusters in some areas, and flat sheets of cells or even single cells in others. By 24 h, the biofilm is developed in the control catheters. If the goal is to monitor early stage biofilm development, then tubing and effluent samples need to be collected prior to the 24 h sample collection. Monitoring biofilm development requires sampling. The biofilm generated in the Intraluminal Catheter Model is suitable for comparison testing between antimicrobial and control catheters.
SCOPE
1.1 This test method specifies the operational parameters required to assess the ability of antimicrobial urinary catheters to prevent or control biofilm growth. Efficacy is reported as the log reduction in viable bacteria when compared to a repeatable (1)2  Escherichia coli biofilm grown in the intra-lumen of a urinary catheter under a constant flow of artificial urine.  
1.2 The test method is versatile and may also be used for growing and/or characterizing biofilms and suspended bacteria of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism.  
1.3 This test method may be used to evaluate surface modified urinary catheters that contain no antimicrobial agent.  
1.4 This test method describes how to sample and analyze catheter segments and effluent for viable cells. Biofilm population density is recorded as log colony forming units per surface area. Suspended bacterial population density is reported as log colony forming units per volume.  
1.5 Basic microbiology training is required to perform this test method.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-2021
• ICS: 07.100.01 - Microbiology in general
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jul-2020
• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM D5465-16 - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jun-2016
• Refers: ASTM D5465-93(2012) - Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jun-2012
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM D5465-93(2004) - Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jun-2004
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002
• Refers: ASTM D5465-93(1998) - Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 10-Dec-1998

Overview

ASTM E3321-21 specifies a standardized laboratory test method for evaluating the performance of antimicrobial urinary catheters in preventing or controlling Escherichia coli (E. coli) biofilm growth within the catheter lumen. Medical device-associated infections, particularly catheter-associated urinary tract infections (CAUTIs), remain a significant healthcare challenge. Biofilm formation by bacteria such as E. coli is a primary cause of persistent device-related infections, as biofilms can be highly tolerant to antibiotics. This method utilizes the Intraluminal Catheter Model (ICM) under continuous flow of artificial urine to simulate clinical conditions and provides a repeatable means of measuring the efficacy of urinary catheters with or without antimicrobial modifications.

Key Topics

• Biofilm Formation: Biofilm is a structured community of bacteria adhered to surfaces, such as urinary catheters. It is a critical factor in the development of persistent infections due to its resistance to antibiotic treatments.
• Antimicrobial Efficacy: The standard provides guidance on evaluating whether urinary catheters, either coated with antimicrobial agents or modified surfaces, effectively inhibit E. coli biofilm growth compared to untreated controls.
• Test Procedures:
  • Standardized inoculation and growth of E. coli in a catheter using artificial urine under constant flow.
  • Sampling and analysis of catheter segments and effluent for viable bacterial counts.
  • Calculation of log reduction in viable colony forming units (CFU) as the primary measure of antimicrobial performance.
• Versatility: While focused on E. coli, the test method may be adapted for other microbial species by adjusting operational parameters as needed.
• Data Reporting: Presents results as log reduction of CFU, supporting quantitative comparison between test catheters.

Applications

• Healthcare Product Evaluation: Manufacturers and researchers use ASTM E3321-21 to assess the effectiveness of antimicrobial urinary catheters before regulatory submission or market release.
• Product Development: Supports the optimization of surface coatings and antimicrobial agents by providing reliable, reproducible test conditions.
• Clinical Risk Reduction: Helps in selecting urinary catheters that minimize biofilm formation, thus reducing the risk of CAUTI and associated hospital-acquired infections.
• Comparative Testing: Enables side-by-side performance assessment of newly developed antimicrobial catheters versus standard uncoated devices.
• Regulatory Compliance: Provides verifiable laboratory data often required by health authorities for product approval or labeling related to infection prevention.

Related Standards

• ASTM D5465 – Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• ASTM E1054 – Practices for Evaluation of Inactivators of Antimicrobial Agents
• ASTM E2756 – Terminology Relating to Antimicrobial and Antiviral Agents
• Other Standards in Infection Control:
  • Standards addressing in vitro assessment of medical device coatings for antimicrobial activity
  • Guidelines for preventing CAUTI in healthcare facilities

Practical Value

ASTM E3321-21 plays a crucial role in infection control and patient safety by providing a validated and transparent laboratory method for determining the microbiological safety and effectiveness of urinary catheters. Through the accurate simulation of urinary tract conditions and precise quantification of biofilm development, this method helps advance the design and regulatory approval of safer urological devices, ultimately decreasing healthcare-associated infection rates and improving patient outcomes.

Keywords: ASTM E3321-21, antimicrobial urinary catheter, biofilm prevention, Escherichia coli, CAUTI, infection control, intraluminal catheter model, healthcare-associated infection, device testing, artificial urine model.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation:E3321 −21
Standard Test Method for
Intraluminal Catheter Model used to Evaluate Antimicrobial
Urinary Catheters for Prevention of Escherichia coli Biofilm
Growth
This standard is issued under the fixed designation E3321; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
1.1 This test method specifies the operational parameters
Barriers to Trade (TBT) Committee.
required to assess the ability of antimicrobial urinary catheters
topreventorcontrolbiofilmgrowth.Efficacyisreportedasthe
2. Referenced Documents
log reduction in viable bacteria when compared to a repeatable
2 2.1 ASTM Standards:
(1) Escherichia coli biofilm grown in the intra-lumen of a
D5465Practices for Determining Microbial Colony Counts
urinary catheter under a constant flow of artificial urine.
from Waters Analyzed by Plating Methods
1.2 The test method is versatile and may also be used for
E1054PracticesforEvaluationofInactivatorsofAntimicro-
growing and/or characterizing biofilms and suspended bacteria
bial Agents
of different species, although this will require changing the
E2756Terminology Relating toAntimicrobial andAntiviral
operational parameters to optimize the method based upon the
Agents
growth requirements of the new organism.
3. Terminology
1.3 This test method may be used to evaluate surface
3.1 Definitions: For definitions of terms used in the test
modified urinary catheters that contain no antimicrobial agent.
method see Terminology E2756.
1.4 This test method describes how to sample and analyze
catheter segments and effluent for viable cells. Biofilm popu-
4. Summary of Test Method
lation density is recorded as log colony forming units per
4.1 This test method is used for evaluating the growth of
surface area. Suspended bacterial population density is re-
Escherichia coli ATCC 53498 biofilm in an antimicrobial
ported as log colony forming units per volume.
Foley catheter.Antimicrobial catheters are tested in parallel to
1.5 Basic microbiology training is required to perform this
control catheters. Prior to inoculation with an overnight
test method.
culture, sterile artificial urine media (AUM) is flowed through
the catheter for 2 h. During the entire test, the biofilm is
1.6 The values stated in SI units are to be regarded as
exposed to a continuous, slow flow ofAUM through the Foley
standard. No other units of measurement are included in this
catheter. At the end of each 24 h time period, bacteria are
standard
quantified by collecting 10 mL of the effluent anda2cm
1.7 This standard does not purport to address all of the
segment from the distal end of the catheter. The biofilm is
safety concerns, if any, associated with its use. It is the
harvested by scraping the lumen of the catheter segment into
responsibility of the user of this standard to establish appro-
10 mL neutralization broth then placing the scraped segment
priate safety, health, and environmental practices and deter-
intotheneutralizationbrothtobeprocessed.Botheffluentflow
mine the applicability of regulatory limitations prior to use.
andbiofilmsamplesarevortexedandsonicatedtodisaggregate
1.8 This international standard was developed in accor-
the clumps and diluted and plated for viable cell enumeration.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
5. Significance and Use
5.1 In the battle to reduce medical device and implant-
This test method is under the jurisdiction of ASTM Committee E35 on
related infections, prevention of bacterial colonization of
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2021. Published November 2021. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E3321–21. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3321−21
surfacesisalogicalstrategy.Bacterialcolonizationofasurface at least two 1 cm holes bored in the same fashion (one for
is a precursor to biofilm formation. Biofilm is the etiological effluent waste and one for bacterial air vent).
agent of many implant and device-related infections and once
NOTE 1—Carboy tops can be purchased with fittings.
established,microorganismsinbiofilmcanbeupto1000times
6.12 Chemical Spatulas.
more tolerant to antibiotic therapy. Often the best treatment
strategy is removal of the implant or device at a high
6.13 Clamp Stand—Height no less than 76.2 cm, used with
socioeconomic cost. Catheter associated urinary tract infec-
clamp to suspend glass flow break vertically and stabilize
tions (CAUTI) are the most prevalent of the device-related tubing.
healthcare associated infections. Catheter associated infections
NOTE 2—If a clamp stand does not fit in the incubator, tape may be
account for 37% of all hospital acquired infections (HAI) and
used.
70% of all nosocomial urinary tract infections (UTI) in the
6.14 Colony Counter—Any one of several types may be
U.S. (2, 3). The Intraluminal Catheter Model (ICM) was
used. A hand tally for the recording of the bacterial count is
developed to evaluate the ability of antimicrobial catheters to
recommended if manual counting is done.
inhibit biofilm growth on the catheter lumen.
6.15 Conical Centrifuge Tubes—Sterile, any with 15mL
5.2 Thepurposeofthistestmethodistodirectauserinhow
and 50 mL volume capacity.
to grow, sample, and analyze an E. coli biofilm in a urinary
catheter under a constant flow of artificial urine. The test 6.16 Culture Tubes and Culture Tube Closures—Any with a
volumecapacityof10mLandaminimumdiameterof16mm.
method incorporates operational parameters utilized in similar
published methods (4). The E. coli biofilm that grows has a Recommended size is 16mm by 125 mm borosilicate glass
with threaded opening.
patchy appearance that varies across the catheter.
Microscopically, the biofilm is heterogenous, with large clus-
6.17 Disposable Bottle Top Filtration Units and 1 L Re-
ters in some areas, and flat sheets of cells or even single cells
ceiver Flasks—sterile, nylon filter with 0.2 µm pore size.
in others. By 24 h, the biofilm is developed in the control
6.18 Ethanol, 95 %—Used to flame sterilize hemostats or
catheters. If the goal is to monitor early stage biofilm
forceps.
development, then tubing and effluent samples need to be
collected prior to the 24 h sample collection. Monitoring 6.19 Glass Flow Breaks—Any that will connect with tubing
biofilm development requires sampling. The biofilm generated of ID 3.1 mm and withstands sterilization.
in the Intraluminal Catheter Model is suitable for comparison 6.19.1 Clamp—Used to hold flow breaks, extension clamp
testing between antimicrobial and control catheters. with 0.5 cm minimum grip size.
6.20 Incubator—that can maintain a temperature of 36°C -
6. Apparatus
38°C.
6.1 Aluminum foil.
6.21 Inoculating Loop—10 µL.
6.2 Analytical Balance—Sensitive to 0.01 g.
6.22 Magnetic Stir Plate, Heated—Used to dissolve chemi-
cals in stock solutions.
6.3 Applicator Sticks, wooden—sterile.
6.23 Norprene Tubing—size 16 with ID 3.1 mm and outer
6.4 Bacterial Air Vent (Filter)—autoclavable, 0.2 µm pore
diameter (OD) 3.2 mm. Must withstand sterilization.
size.
6.24 Peristaltic Pump—One pump head capable of holding
6.5 Barbed Bulkhead Fittings—autoclavable, plastic, inner
tubingwithID3.1mmandOD3.2mmandoperatingataflow
diameter (ID) 0.25 in.
rate of 0.75 mL per minute.
6.6 Biosafety Cabinet.
NOTE 3—A digital pump, or pump with high sensitivity will be
6.7 Bacterial Air Vent (Filter)—Autoclavable 0.2 µm pore
beneficial for maintaining a low flow rate. Pump must be calibrated
size, to be attached into tubing on waste and nutrient carboy
following the manufacturer’s recommendations using the same tubing
configuration that will be utilized in the experiment.
(recommended diameter is 37 mm) and each reactor channel
top (recommended diameter is 15 mm).
6.25 Petri Dish—100mm by 15 mm, plastic, sterile, and
empty for transporting samples from the incubator to the
6.8 Bacterial Air Vent (Filter)—autoclavable, 0.2 µm pore
workstation.
size.
6.26 pH Meter and Electrode—Used for adjusting pH of
6.9 Barbed Bulkhead Fittings—autoclavable, plastic, ID
AUM.
0.25 in.
6.27 Pipettes—Serological sterile single-use pipettes with
6.10 Bunsen Burner—Used to flame sterilize inoculating
volume capacity of 5 mL, 10 mL, 25 mL, and 50 mL.
loop and other instruments.
6.28 Pipettor—Continuouslyadjustablepipettewithvolume
6.11 Carboys—One 5 L glass and one 20 L autoclavable
capability of 1 mL.
carboy for nutrients and waste, respectively.
6.11.1 Carboy Lids—One 5 L glass carboy lid with at least
twobarbedfittingstoaccommodatetubingID3.1mm(onefor
nutrientlineandoneforbacterialairvent).Onecarboylidwith Trademarked by the Saint-Gobain Performance Plastics Corporation.
E3321−21
6.29 Ruler—Used to measure the correct length of catheter 6.43 Water Bath, Ultrasonic—–any capable of maintaining
tubing. a homogeneous sound distribution at 45kHz 6 5kHz with a
volume large enough to accommodate 50 mL conical tubes in
6.30 Silicone Tubing—Three sizes of silicone tubing: size
a wet environment.
16 with ID 3.1 mm and OD 3.2 mm, size 18 with ID 7.9 mm
and OD 9.5 mm, size 25 with ID 4.8 mm and OD 4.9 mm.All
NOTE 4—Verify that the sonicating bath does not kill viable cells by
placing the standardized broth culture into sonicator for 60s, serially
sizes must withstand sterilization.
dilute,andplate.Thesonicatedandnon-sonicatedcountsshouldbewithin
6.31 Size 16 French Foley catheter—Sterile, silicone.Anti-
0.5 logs.
microbial and control catheters that are 30 cm in length with
6.44 Weigh Boats or Papers.
the same inner diameter should be used.
6.32 Stainless Steel Scissors—Forasepticcuttingofcatheter
7. Reagents and Materials
tubing.
7.1 Purity of Water—All reference to water as diluent or
6.33 Stainless Steel Hemostat Clamp or Forceps—Forasep-
reagent shall mean distilled water or water of equal purity.
tic handling of catheter tubing.
7.2 Culture Media:
6.34 Sterilizers—Any steam sterilizer capable of producing
7.2.1 Bacterial Liquid Growth Broth—Artificial Urine Me-
the conditions of sterilization.
dium (5) is required.
6.35 Stir Bars, Magnetic—Used to dissolve chemicals in
7.2.1.1 Solution 1—1.85g CaCl ×2H O, 26.0g NaCl,
2 2
stock solutions.
2.45g MgSO ×7H O, 8.45g Na SO,6.5gNH Cl, 5g
4 2 2 4 4
peptonein4580mLdeionizedwaterin5Lglassvessel,mixed
6.36 Straight Tubing Connectors—ID 3.1 mm and ID 7.9
withamagneticstirbarandstirplate,autoclavedandcooledto
mm. Must withstand sterilization.
room temperature.
6.37 System Components—A schematic of the tubing from
7.2.1.2 Solution 2—0.5 g/L yeast extract, 5 g/L lactic acid,
the AUM carboy to where the tubing connects to the catheter
40 g/L citric acid in distilled water, mixed and filter sterilized.
is shown in Fig. 1. The Intraluminal Catheter Model is shown
7.2.1.3 Solution 3—25 g/L creatinine, 0.06 g⁄L
in Fig. 2.
FeSO ×7H O in distilled water, mixed and filter sterilized.
4 2
6.38 Three way Stop-Cock Valve—4mm and autoclavable.
7.2.1.4 Solution 4—95 g/L KH PO in distilled water,
2 4
mixed and filter sterilized.
6.39 Timer—Used to time the effluent collection.
7.2.1.5 Solution 5—120 g/L K HPO in distilled water,
2 4
6.40 Tubing, Silicone—ID 3.1 mm, size 16, OD 3.2 mm.
mixed and filter sterilized.
6.41 Vacuum source—in-house line or suitable vacuum
7.2.1.6 Solution 6—400 g/L urea in distilled water, mixed
pump for filtering.
and filter sterilized.
6.42 Vortex—Any vortex that will ensure proper agitation 7.2.1.7 Solution 7—105 g/L NaHCO in distilled water,
and mixing of culture tubes. mixed and filter sterilized.
FIG. 1Tubing Set-up from the AUM Carboy to Where the Tubing Connects to the Catheter
E3321−21
FIG. 2Intraluminal Catheter Model
7.2.1.8 Solution 8—83 mL/L HCl in distilled water, mixed 8.1.2 Attach a length of silicone tubing to the underside of
and filter sterilized. two of the bulkhead fittings to reach the bottom of the vessel.
7.2.2 Bacterial Plating Medium—Tryptic Soy Agar (TSA )
8.1.3 Attach short pieces of tubing to the top of all three
is required.
bulkhead fittings. Fit a tubing connector to the two with tubing
interior to the vessel. Fit a bacterial air vent to the remaining
7.3 Buffered Water—0.0425 g/L KH2PO4 distilled water,
piece of tubing on the top of the vessel lid.
filter sterilized and 0.405 g/LMgCl·6H O distilled water, filter
8.1.4 Cover the two connectors and interior tubing with
sterilized (prepared according to Method 9050 C.1a).
aluminum foil. Leave the bacterial air vent uncovered.
7.4 Neutralizer—Dey/Engley Neutralizing Broth or one
8.1.5 Place the lids in an autoclavable container and steril-
specific to the antimicrobial being evaluated as determined for
ize.
effectiveness and toxicity according to Practices E1054 in
accordance with Section 8 or Section 10. Conduct neutra
...