WO2000077162A1 - Biofilm modele pour l'evaluation de l'efficacite d'agents antimicrobiens - Google Patents

Biofilm modele pour l'evaluation de l'efficacite d'agents antimicrobiens Download PDF

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Publication number
WO2000077162A1
WO2000077162A1 PCT/US2000/015675 US0015675W WO0077162A1 WO 2000077162 A1 WO2000077162 A1 WO 2000077162A1 US 0015675 W US0015675 W US 0015675W WO 0077162 A1 WO0077162 A1 WO 0077162A1
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WO
WIPO (PCT)
Prior art keywords
biofilm
model
cells
growth medium
coupons
Prior art date
Application number
PCT/US2000/015675
Other languages
English (en)
Inventor
Ursula K. Charaf
Original Assignee
S.C. Johnson & Son, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by S.C. Johnson & Son, Inc. filed Critical S.C. Johnson & Son, Inc.
Priority to AU54702/00A priority Critical patent/AU5470200A/en
Priority to CA002376372A priority patent/CA2376372A1/fr
Priority to BR0011416-2A priority patent/BR0011416A/pt
Priority to EP00939646A priority patent/EP1185614A1/fr
Priority to JP2001503998A priority patent/JP2003502063A/ja
Publication of WO2000077162A1 publication Critical patent/WO2000077162A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates

Definitions

  • Biofilm cells are phenotypically different from planktonics (Characklis, 1990; Gilbert, 1990; Costerton, et al, 1995).
  • One of the most important manifestations of this difference is the significantly decreased susceptibility of biofilm cells to biocides (Costerton, et a ⁇ ., 1995; Das, et __[., 1997).
  • Disinfectant registration protocols are based on the acceptance that microorganisms grown suspended in liquid laboratory cultures are generally representative of those found in the environment.
  • biofilm testing in a disinfectant testing regime would be to substitute biofilm covered carriers for those covered with planktonic cells as used in current tests approved by the Association of Official Analytical Chemists (AOAC).
  • AOAC Association of Official Analytical Chemists
  • Biofilm-covered coupons suitable for disinfectancy testing can be grown in a variety of biofilm reactors. However, these methods require expertise and the use of expensive equipment and produce a limited number of available test coupons.
  • model biofilm that is reliable, simple to prepare and not dependant on expensive equipment.
  • the model biofilm is grown on coupons, or other planar surfaces that are placed on inoculated growth media, such as filter paper, on a nutrative support, such as agar. These biofilm-covered coupons may be substituted for the carriers covered with planktonic cells that are used in conventional biocide testing.
  • the model biofilm is a naturally grown biofilm and the cells are not placed in an artificial environment.
  • the model biofilm is neither "reactor grown” nor grown at a solid/liquid interface, as are most test biofilms.
  • the biofilm of the present invention is grown under controlled conditions and is reproducible.
  • One embodiment of the invention comprises the steps of placing a plurality of surfaces on an inoculated growth medium (wherein the growth medium contacts a nutrative source), growing a model biofilm on the bottom of the plurality of surfaces, and removing the surfaces from the growth medium.
  • the surfaces will be covered with the model biofilm of the present invention. It is an object of the present invention to provide a plurality of reproducible test surfaces covered with model biofilm for use in testing protocols. It is another object of the present invention to provide a rapid, reproducible, and low cost method for creation of model biofilm test surfaces.
  • Fig. 1 is a schematic of one embodiment of a model biofilm set up.
  • Fig. 2 is a bar graph recording the number of cells recovered from different control runs.
  • Fig. 3 is a bar graph describing the effect of hypochlorites on model biofilms of the present invention.
  • Fig. 4 is a bar graph describing the effect of test products on biofilm.
  • Fig. 5 is a bar graph tabulating the effect of two test biocides on three different types of microorganisms, e.g. Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae.
  • Fig. 6 is a bar graph comparing changes in inoculum and nutrient availability in a Staphylococcus aureus model biofilm.
  • Fig. 7 is a bar graph comparing cell numbers recovered from model biofilm of the present invention versus reactor grown biofilm.
  • the cells of the biofilm of the present invention are embedded in EPS that the cells produce. I prefer to categorize this biofilm as a 'model' because I do not know that the cells have undergone ajl of the prerequisite phenotypic changes attributed to a true biofilm.
  • the model biofilm does have some of the characteristics of wild biofilms. For example, the cells adhere to glass slides or other test surfaces, produce slime, and show significantly increased resistance to antimicrobial agents.
  • attached cells with visible slime production are in a range of 10 3 to 10 12 cells per cm 2 . The preferred range is 10 7 -10 8 . It is preferable that the solid coupon does not directly touch a solid, non-porous surface.
  • a model biofilm may be validated against those obtained from natural or reactor-grown biofilm samples that are treated identically.
  • Method for preparing model biofilm in one embodiment, disclosed in Fig. 1 , the method involves growing biofilm 2 on an inoculated growth medium 4, preferably filter paper (Whatman qualitative #2), placed on top of a nutrative source 6, preferably agar, e.g. Trypticase Soy Agar. I used 10 x 10 cm square Petri dishes with 40 ml agar per plate.
  • Suitable nutrative sources are those that can support a porous sheet. This could be a liquid medium with a frame or other structure supporting the filter paper or a sponge-like sheet saturated with medium (i.e., the porous support could be thick enough to make the agar underneath unnecessary). Therefore, the nutrative source and the inoculated growth medium may be the same physical structure.
  • a quantity, usually 1 ml, of a diluted (1/10 to 1/100) overnight culture of a desired biofilm-forming organism is pipetted onto the filter paper so that the entire paper surface is evenly moistened.
  • Filter paper is porous, and its purpose is to reduce the possibility of anoxic conditions developing on the underside of the coupons.
  • porous growth media may be suitable. It is only necessary that the suitable growth media support the growth of the test organisms.
  • sterile flat coupons e.g. glass or stainless steel
  • the coupons are aseptically removed from the surface of the filter paper with a forceps and used either immediately or after drying for 40 minutes at 35 ⁇ 2°C.
  • the drying step corresponds to the prior art preparation of planktonic carriers.
  • the coupons are then subjected to biocide testing, preferably as outlined in AOAC Official Methods of Analysis.
  • EPS extracellular polymeric substance
  • planktonics qualitative. Each active ingredient was first tested on planktonic cells following the procedure outlined in the AOAC Germicidal 0 Spray test. A cell suspension was applied to flat glass coupons, 6.44 cm 2 in size. The coupons were dried for 40 minutes at 35 ⁇ 2°C and sprayed with a biocidal treatment. After an exposure time of 10 minutes, the coupons were transferred to a neutralizing broth, incubated for 48 hours and observed for growth. According to the AOAC Official Method, an active or product passes 5 the disinfectancy test if no more than 1 out of 60 tubes shows growth due to surviving cells.
  • R2A media is supplied by BBL or Difco (DF1826-17-1 ), included in Handbook of Microbiological Media, Ronald M. Atlas, CRC Press, 1993, ed. Lawrence C. Parks. Cells surviving the treatment were counted as o CFU's after 24 to 48 hours of incubation. An equal number of untreated coupons was also scraped into the neutralizing broth and processed identically to the treatments. These served as controls used to calculate log reductions by a method developed at the Center for Biofilm Engineering at Montana State University (Hamilton and Herigstad, 1998).
  • C. Results/Discussion The advantage of this model biofilm is its ease of preparation and reproducibility between samples.
  • the cell density of a 48-hour model biofilm consistently reached about 10 8 cells/coupon with variability within 1 log (Fig. 2).
  • the model biofilm can easily be seen with the naked eye as a slimy material adhering to the underside of the coupons. Microscopic examination by brightfield microscopy shows cell clusters associated with EPS. In fact, our model biofilm is indistinguishable from stained reactor grown biofilm, although confocal microscopy may reveal differences in biofilm architecture.
  • Biocidal efficacy testing enables the evaluation and ranking of biocidal products.
  • Biofilm coupons were substituted for the planktonic preparations in the AOAC Germicidal Spray Test and other standard antimicrobial test to obtain a qualitative assessment of the disinfectant efficacy of products versus biofilm.
  • initial cell numbers on biofilm and planktonic test coupons were comparable, i.e. 10 8 to 10 9 cells per coupon (10 6 to 10 7 cells/cm 2 of coupon surface). I believe that the results thus obtained are in range with results described in the literature for artificial biofilms (Chen and Stewart, 1996) as well as for environmental or reactor grown biofilms (Samrakandi, et aj., 1997).
  • Table 1 shows the results for the qualitative AOAC Germicidal Spray tests for Pseudomonas aeruginosa PA01 and Staphylococcus aureus 6538 planktonic cells and biofilm. All tests involving planktonics consistently passed the AOAC Germicidal Spray Test, i.e. there were no surviving cells on any of the 60 coupons that had been treated. In contrast, none of the identically treated biofilm samples passed. Pseudomonas aeruginosa Staphylococcus aureus
  • Table 1 AOAC qualitative Germicidal Spray Test: Effect of biocides on planktonic cells and biofilm.
  • Staphylococcus aureus ATCC 6538
  • Klebsiella pneumoniae Enterobacter aerogenes against 2 standard test biocides (see Fig. 5).
  • Staphylococcus aureus showed slightly higher kill in response to both biocides than
  • Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes almost identically showed reduction of cells of only approximately 1 log or less. It may be advantageous to eventually tailor biofilm composition for test applications according to the predominant species found in the respective environment. Little is known about the synergistic or antagonistic actions of mixed biofilms with respect to biocides and the method of the present invention may be a suitable tool for this evaluation.
  • Fig. 6 is a bar graph describing the results with the Staphylococcus aureus model.
  • Staphylococcus aureus decreasing the number of cells placed on the porous medium did not change the number of biofilm cells harvested after 48 hours. Decreasing the nutrient concentration of the agar only affected the number of biofilm cells significantly below 5% (i.e. at 1 %) of the original nutrient concentration.
  • Klebsiella pneumoniae a similar trend was observed. (Empty boxes represent experiments not done because a trend can be established from the available data.)
  • the model biofilm described above was exposed to two stains: Alcian Blue for polysaccharide and Carbol Fuchsin for cells. I stained all cell types, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter aerogenes. All preparations showed cells associated with EPS. The clustering effect was most prominent for Staphylococcus aureus, which has a natural trend to form microcolonies quickly. In this way, the polysaccharide was stained blue and the cells red. I showed clearly that polysaccharide was present and that it was closely associated with the cells. In particular, Staphylococcus aureus model biofilm cells were observed to cluster into microcolonies, such biofilm cells are described in the literature. F. Comparison of model biofilm with reactor-grown biofilm.
  • Table 4 and Fig. 7 describe the comparison of model biofilm with reactor- grown biofilm.
  • This treatment data show that the model biofilm reacts to the standard treatments, i.e. 1000 ppm NaOCI and 1000 ppm Quat + 200 ppm EDTA, in a manner similar to reactor-grown biofilm.
  • the modle biofilm of the present invention provides a multitude of reproducible test surfaces for use in testing protocols. Preparing the modle antimicrobial biofilm involves relatively inexpensive equipOment and materials and is not limited by the number of test coupons. The method is fast (48 hours), simple and reproducible.

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  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Cette invention se rapporte à un procédé servant à faire croître un biofilm modèle. Dans un mode de réalisation, ce procédé consiste à placer plusieurs surfaces comprenant une partie supérieure et une partie inférieure sur un support de croissance inoculé, ce support de croissance étant en contact avec une source nutritive, à faire croître un biofilm modèle sur la partie inférieure de ces surfaces, et à retirer lesdites surfaces du support de croissance, de telle sorte qu'un biofilm modèle se trouve déposé sous la forme de revêtement sur la partie inférieure de ladite surface.
PCT/US2000/015675 1999-06-10 2000-06-07 Biofilm modele pour l'evaluation de l'efficacite d'agents antimicrobiens WO2000077162A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU54702/00A AU5470200A (en) 1999-06-10 2000-06-07 A model biofilm for efficacy assessment of antimicrobials
CA002376372A CA2376372A1 (fr) 1999-06-10 2000-06-07 Biofilm modele pour l'evaluation de l'efficacite d'agents antimicrobiens
BR0011416-2A BR0011416A (pt) 1999-06-10 2000-06-07 Biofilme-modelo para a avaliação da eficácia de antimicrobianos
EP00939646A EP1185614A1 (fr) 1999-06-10 2000-06-07 Biofilm modele pour l'evaluation de l'efficacite d'agents antimicrobiens
JP2001503998A JP2003502063A (ja) 1999-06-10 2000-06-07 殺菌剤の効力評価用モデルバイオフィルム

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13835499P 1999-06-10 1999-06-10
US60/138,354 1999-06-10

Publications (1)

Publication Number Publication Date
WO2000077162A1 true WO2000077162A1 (fr) 2000-12-21

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EP (1) EP1185614A1 (fr)
JP (1) JP2003502063A (fr)
AR (1) AR024565A1 (fr)
AU (1) AU5470200A (fr)
BR (1) BR0011416A (fr)
CA (1) CA2376372A1 (fr)
WO (1) WO2000077162A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022245A1 (fr) * 2002-09-06 2004-03-18 The Secretary Of State For Defence Procede d'inoculation et appareil a cet effet
WO2004087894A2 (fr) * 2003-04-01 2004-10-14 S.C.Johnson & Son, Inc. Agregats bacteriens mis au point
EP2229959A2 (fr) 2009-03-18 2010-09-22 EMPA Eidgenössische Materialprüfungs- und Forschungsanstalt Production standardisée de biofilms matures

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7370203B2 (ja) * 2019-09-24 2023-10-27 小林製薬株式会社 黒ずみ形成方法
WO2021182121A1 (fr) * 2020-03-13 2021-09-16 学校法人慈恵大学 Réactif de mise en transparence de biofilm, et procédé d'observation de biofilm mettant en œuvre ledit réactif de mise en transparence

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4125436A (en) * 1977-08-25 1978-11-14 Linbro Scientific, Inc. Slips for specimen growth and microscopic examination
WO1991001364A1 (fr) * 1989-07-24 1991-02-07 Imperial Cancer Research Technology Limited Dispositif de transfert d'echantillons
WO1995027039A1 (fr) * 1994-03-31 1995-10-12 Kemira Chemicals Oy Dispositif d'examen de film biologique destine a la surveillance de perturbations microbiennes dans des eaux de traitement industriel

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4125436A (en) * 1977-08-25 1978-11-14 Linbro Scientific, Inc. Slips for specimen growth and microscopic examination
WO1991001364A1 (fr) * 1989-07-24 1991-02-07 Imperial Cancer Research Technology Limited Dispositif de transfert d'echantillons
WO1995027039A1 (fr) * 1994-03-31 1995-10-12 Kemira Chemicals Oy Dispositif d'examen de film biologique destine a la surveillance de perturbations microbiennes dans des eaux de traitement industriel

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022245A1 (fr) * 2002-09-06 2004-03-18 The Secretary Of State For Defence Procede d'inoculation et appareil a cet effet
WO2004087894A2 (fr) * 2003-04-01 2004-10-14 S.C.Johnson & Son, Inc. Agregats bacteriens mis au point
WO2004087894A3 (fr) * 2003-04-01 2005-03-24 Johnson & Son Inc S C Agregats bacteriens mis au point
EP2229959A2 (fr) 2009-03-18 2010-09-22 EMPA Eidgenössische Materialprüfungs- und Forschungsanstalt Production standardisée de biofilms matures

Also Published As

Publication number Publication date
JP2003502063A (ja) 2003-01-21
AR024565A1 (es) 2002-10-16
CA2376372A1 (fr) 2000-12-21
AU5470200A (en) 2001-01-02
EP1185614A1 (fr) 2002-03-13
BR0011416A (pt) 2002-03-26

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