University of Groningen

Synthesis of quaternary ammonium coated surfacesRoest, Steven

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Chapter 6

Comparison of methods to evaluate bacterial contact-killing

on cationic surfaces

Roest, S.; Loontjens, T. J. A.; Busscher, H. J., Van der Mei, H. C.;

To be published.

Chapter 6

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6.1 Abstract

Prevention of implant related infections by the design of antimicrobial coatings on biomaterials

implants and devices has attracted an increasing amount of interest. Cationic contact-killing

coatings constitute a special class of antimicrobial coatings. Since they do not rely on release of

antimicrobials but only act upon contact with adhering bacteria, they offer several advantages above

release coatings. However, reliable in vitro evaluation methods for bacterial contact-killing surfaces

do not yet exist, while more importantly results of different evaluation methods are often

conflicting. The aim of this study was to compare five methods to evaluate bacterial contact-killing

surfaces. Our comparison is based on determining the contact-killing efficacy of an established,

contact-killing alkylated hyperbranched polyurea-polyethyleneimine coating upon contact with a

Staphylococcus epidermidis strain. Depending on the method used, different results were obtained in

bacterial contact-killing. We conclude that the Petrifilm® and Japanese Industrial Standards (JIS)

methods are preferable: Petrifilm® is most convenient and possibly more reliable. Like all others,

these methods need a complementary assay to exclude killing resulting from release of

antimicrobial compounds, because even a small release of an antimicrobial compound will have a

large influence on bacterial killing in the small fluid volumes of the assays. The modified JIS method

is acceptable, but does not contain balanced amount of nutrients compared to the Petrifilm®

method and should only be used with respect to a non-contact killing control. ASTM and bacterial

spray methods are not reliable, the main reason being the lack of control over the applied bacterial

challenge.

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6.2 Introduction

Bacterial adhesion and subsequent biofilm formation on surfaces of biomaterials implants or

devices such as total hip or knee arthroplasties and catheters, constitute the major cause of implant

or device failure.[1-3] These biomaterial-associated infections (BAI) are difficult to treat with

antibiotics as a result of the protection offered by the biofilm mode of growth and a reduced

efficacy of the host immune system around a biomaterial implant or device.[4-6] Moreover, bacteria

may seek shelter in tissue surrounding biomaterial implants and devices.[7] Initially, clinicians will

try to treat patients suffering from BAI with antibiotics, but often the outcome is revision surgery,

accompanied by great discomfort for the patient[8-9] and treatment costs that on average triple the

costs of the primary implant or device.[10] The lack of antibiotic efficacy and the anticipated growth

in the number of biomaterial implants and devices worldwide[11] increased the development of new

antibacterial or infection-resistant biomaterials.[12-14] However, their downward translation to

clinical use fails for a variety of highly diverse reasons,[2,15] including lack of proper evaluation

methods.

The different coatings that are being considered as antibacterial or infection-resistant are either

non-adhesive to bacteria such as hydrophobic coatings,[16-17] polyethylene glycol (PEG) brush

coatings,[18-19] hydrogel coatings,[20] coatings with nanoparticles[21] or antimicrobial releasing

coatings,[22] which are aimed to yield high particle or antibiotic concentrations around an implant

or device in order to kill the bacteria present.[23] As a drawback of antimicrobial release coatings,

they all show a high-burst release immediately upon insertion in the human body, followed by an

extensive tail-release that can extend to several years and that has been associated with the

development of antibiotic-resistant strains.[24-25]

Cationic coatings possess the unique feature of killing bacteria upon contact [12,26]. Provided the

cationic charge density is above 1014 positive charges per cm2,[27-28] cationically-charged coatings will

kill bacteria upon contact while still facilitating tissue integration. In vivo efficacy of cationic coatings

has been demonstrated in rats[29] and sheep.[30] This negates a common criticism toward cationic

coatings that in the human body layers of dead bacteria and adsorbed proteins will hamper bacterial

contact-killing in vivo. Bacterial debris is indeed cleared by macrophages, although phagocytosis of

adhering staphylococci on a cationically-charged coating was found to be reduced compared with

common, negatively-charged biomaterials.[31] Bacteria adhering to adsorbed protein layers were

equally killed as when adhering to a bare cationically-charged coating,[32] possibly because bacteria

adhering to a protein film slowly sink through the layer[33] to eventually contact the positive charge

facilitating their own death.

No ubiquitously accepted method to evaluate the efficacy of bacterial contact-killing of cationic

surfaces exist. Often applied methods (see also Table 6.1) include the ASTM (American Society

for Testing and Materials),[34] the JIS (Japanese Industrial Standards)[35] and the modified JIS

method,[36] spray-coating of bacteria on a surface[37] and the Petrifilm® assay.[38] A comparison of

methods to establish bacterial contact-killing on cationic surfaces has never been made however.

Therefore the aim of this study is to compare the above five methods with respect to their efficacy

to facilitate bacterial contact-killing of a Gram-positive Staphylococcus epidermidis strain. As a contact-

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killing, cationic surface we used a hyperbranched, alkylated polyethyleneimine coating, with

demonstrated ability to facilitate contact-killing of a variety of different bacterial strains in absence

of leaching antimicrobial compounds (see Figure 6.1 and reference 39).

Table 6.1 Summary of methods applied to compare the efficacy of bacterial contact-killing upon adhesion to a cationic surface.

Method Description Advantages Disadvantages References

Bacterial spray

Spraying of a bacterial suspension onto a surface.

Simple concept. Flexible with regards to size of sample.

Difficult to quantify. Bacteria are partly dehydrated by a drying step.

[14,37,49-50]

ASTM E2149

Incubation of a surface in a bacterial suspension while shaking.

Flexible with regards to shape and size of substratum.

An unknown fraction of the bacteria come into contact with the substratum through diffusion.

[46-47,51-54]

JIS Z 2801

Bacteria are contacted with a surface and covered with Parafilm® for a defined time period.

Fast and reproducible. No nutrient availability during the experiment.

[44,55-56]

Modified JIS

Inoculated filters are placed on a contact-killing surface, with or without the addition of serum.

Fast and reproducible. Nutrient availability during the experiment. Proteinaceous conditioning film is applied.

Bacteria might reside deeper in the filter and not come in contact with the coating.

[36,57]

Petrifilm®

Bacteria are confined between a thin nutrient agar layer and a contact-killing surface.

Bacteria are in direct contact with the surface. In situ enumeration.

High numbers of bacteria cannot be counted.

[58-59]

Figure 6.1 Absence of an inhibition zone around an alkylated hyperbranched polyurea-polyethyleneimine coated glass slide on an agar plate, after 96 h incubation with S. epidermidis ATCC 12228, indicating absence of leaching of antibacterial compounds. Taken from Chapter 4.

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6.3 Materials and Methods

6.3.1 Bacterial strains, growth and harvesting

S. epidermidis ATCC 12228, originating from blood of a patient with an intravascular catheter

infection was used in this study. The strain was first streaked on a blood agar plate from a frozen

stock solution (7 v/v% DMSO) and grown overnight at 37°C. One colony was inoculated in 10

mL of tryptone soya broth (TSB, Oxoid, Basingstoke, UK) and incubated at 37°C for 24 h. 10 mL

of this culture was used to inoculate a main culture of 200 mL of TSB, which was incubated for 16

h at 37°C. Bacteria were harvested by centrifugation for 5 min at 6500 g and 10°C and subsequently

washed two times with phosphate-buffered saline (PBS; 10 mM potassium phosphate and 150 mM

NaCl, pH 7.0). Bacterial suspensions were prepared as described above, unless stated otherwise

and concentrations adjusted by dilution to the requirements of the specific methods, while

enumerating the number of bacteria in suspension using a Bürker-Türk counting chamber. The

percentage live bacteria in a suspension was determined by plate counting in triplicate.

6.3.2 Preparation and alkylation of hyperbranched polyethylenimine (PEI) coatings

AB2 monomers, consisting of a secondary amino (A) group and two blocked isocyanate (B) groups

separated by hexyl spacers and the corresponding hyperbranched polymer coatings were prepared

as described before.[40] Glass slides were activated with a piranha solution and subsequently

functionalized with 2-oxo-N-[3-(triethoxysilyl)propyl]-1-azepanecarboxamide as a coupling agent.

The pure hyperbranched polymer was obtained by precipitation in cold water and the isolated

polymer was dried under reduced pressure at 40°C. The functionalized glass slides were submerged

in a solution of 5 wt% hyperbranched polymer and subsequently spinned at 2000 rpm for 60 s.

After annealing, non-anchored polymers were removed by a three-step extraction. First, the

functionalized glass slides were sonicated in ethanol at room temperature (RT) for 20 min, followed

by overnight immersion in dimethylformamide at 115°C, sonication in ethanol at RT for 20 min

and finally dried under nitrogen. A solution of 15 wt% PEI in methanol (800 µL) was dropped on

the hyperbranched coating and spin coated. The grafting reactions were carried out on an

aluminum plate heated to 125°C for 52 h under nitrogen, followed by intermittent sonication in

methanol at RT for 2 x 45 min in fresh solvent each 45 min to extract unreacted components.

Next, PEI functionalized hyperbranched coated glass slides were immersed in 150 mL 1-

bromohexane and heated under nitrogen at 90°C overnight for alkylation. A suspension of 0.6 g

potassium hydroxide powder in 50 mL tert-amyl alcohol was added. The reaction was continued

for another 3 h at 90°C. Afterwards, coated samples were three times sonicated in methanol for 20

min at RT and dried under nitrogen. A second alkylation step was done in a round bottom flask

fitted with a reflux condenser. The coated samples were immersed in a solution of 20 mL

iodomethane in 150 mL tert-amyl alcohol. Alkylation was carried out at 42°C for 18 h.

Subsequently, samples were sonicated in 100 mL methanol for 20 min at RT and followed by

extraction in methanol at 65°C for 1 day and another sonication in methanol for 20 min at RT.

Finally, the QUAT-coated glass slides were dried and stored under nitrogen. Thus prepared coated

samples possess a cationic charge density of 5 x 1016 N+ ions per cm2 and have a water contact

angle of 86.[40] Before use all glass slides with and without (control) a QUAT-coating were cut into

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pieces of 26 x 26 mm (6.76 cm2), sterilized with 70% ethanol and dried in a sterile environment.

These glass slides were used in all experiments, except in the bacterial spray method where slides

of 78 x 26 mm were used.

6.3.3 Methods for evaluating bacterial killing upon contact with a surface

In order to provide for a comparable scale valid for all five methods included in this study, we first

determined the maximum log-reduction in the number of live or viable bacteria that could be

achieved in each method. Next, the actual log-reduction achieved in the method was determined

and expressed as a ratio with respect to the maximal reduction that could have been achieved. This

ratio will be called the “percentage contact-killing efficacy” throughout the remainder of this paper.

6.3.4 Bacterial spray method

This protocol was adapted from Haldar et al.[37] A S. epidermidis ATCC 12228 suspension with a

concentration of 1 x 108 bacteria per mL was sprayed for 3 s onto sterile QUAT-coated or control

glass slides from a distance of approximately 15 cm, placed under an angle of 70 with the

horizontal axis against a wall. After spraying, surfaces with adhering bacteria were dried for 2 min

and stained for 15 min in the dark with 20 µL live-dead Baclight viability stain containing SYTO 9

dye (yielding green fluorescence for live bacteria) and propidium iodide (yielding red fluorescence

in cell membrane-damaged bacteria, generally considered to be “dead” bacteria). Confocal laser

scanning microscopy (CLSM) was used to visualize live and dead adhering bacteria and after

enumeration of the number of live bacteria expressed as a percentage contact-killing efficacy, as

defined above. Experiments were performed in triplicate with three separately grown bacterial

cultures.

6.3.5 ASTM method

In the ASTM E2149 protocol[34] 10 mL of a bacterial suspension (3 x 105 bacteria/mL) was added

into test tubes together with sterile QUAT-coated or control glass slides and shaken in an orbital

mixer at 200 rpm for 15 min. After 5, 10 and 15 min aliquots were taken of the suspension, serially

diluted and the numbers of colony forming units (CFUs) were determined by plate counting on

TSB agar and used to calculate the percentage contact-killing efficacy. In one experiment, aliquots

were taken up to 120 min in order to check whether contact-killing continued after 15 min.

Experiments were performed in triplicate with three separately grown bacterial cultures.

6.3.6 Japanese Industrial Standard method

In the JIS Z 2801:2010,[35] 0.1 mL of a staphylococcal suspension (1 × 106 bacteria/mL) was placed

on sterile QUAT-coated and control glass slides and covered with sterilized Parafilm® (24 × 24

mm2) and subsequently left to incubate at 37°C for 24 h under humidified atmosphere. After

incubation, the Parafilm® was peeled off and placed together with the samples in a small Petri dish

and 5 mL 0.1% (v/v) Tween80 in PBS was added, followed by sonication for 30 s and gentle

shaking for 2 min. The resulting suspensions were serially diluted and the numbers of CFUs were

determined by plate counting on TSB agar from which the percentage contact-killing efficacy was

calculated. Experiments were performed in triplicate with three separately grown bacterial cultures.

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6.3.7 Modified JIS method

In the modified JIS method,[36] 20 µL of a staphylococcal suspension (1 × 107 bacteria/mL) was

pipetted on sterilized nitrocellulose filters (with a pore size of 0.45 µm cut in squares of 24 x 24

mm2) placed on a TSB agar plate. The liquid was absorbed by the agar while the bacteria retained

on the filter. Next, 20 µL of 1% TSB in 10 mM potassium phosphate buffer, with or without 10%

and 50% fetal calf serum added, was pipetted centrally on the surface of sterile QUAT-coated and

control glass slides after which an inoculated filter was carefully placed on top, with the bacteria

contacting the surfaces and left to incubate at 37°C for 24 h in 100% humidified atmosphere. After

incubation, each glass slide and corresponding filter was placed in 5 mL TSB, sonicated for 30 s

and vortexed for 1 min to dislodge adhering bacteria. Finally the resulting suspensions were serially

diluted and the numbers of CFUs were determined by plate counting on TSB agar and employed

to calculate the percentage contact-killing efficacy. Experiments were performed in triplicate with

three separately grown bacterial cultures.

6.3.8 Petrifilm® method

The Petrifilm® Aerobic Count plate system (3M Microbiology, St. Paul, MN, USA) consists of

two films: a bottom film containing standard nutrients, a cold-water gelling agent and an indicator

dye that facilitates colony counting and a top film enclosing the sample within the system. The

bottom film containing the gelling-agent was first swelled with 1 mL sterile demineralized water

for 40 min and transferred to the transparent top film before use. Next, 40 µL of a staphylococcal

suspension (2 x 106 bacteria/mL) was placed on sterile QUAT-coated and control glass slides and

were placed between the two films of the Petrifilm® system and left to incubate at 37°C for 48 h

after which the numbers of CFUs were counted. Closure of the Petrifilm® system ensured

spreading of the staphylococcal suspension over the surface area of the samples. After 48 h, the

number of CFUs on each sample was counted and used to calculate the percentage contact-killing

efficacy. Experiments were performed in triplicate with three separately grown bacterial cultures.

A summary of the methods applied to compare the efficacy of bacterial contact-killing is given in

Table 6.1.

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6.4 Results

The efficacy of bacterial contact-killing by a cationic, QUAT-coated glass slides was investigated

using five different methods, frequently used in literature. Methods were applied as closely as

possible to the instructions given in the literature, which explains why different bacterial challenges

were applied for different methods. Accordingly, the maximal log reduction that could be achieved

differs for each method as can be seen in Table 6.2, variations running from 3.9 log units maximal

reduction for the Petrifilm® assay to 6.8 log units for ASTM E2149.

On control glass slides, none of the methods demonstrated worthwhile contact-killing efficacy of

S. epidermidis ATCC 12228. The spray method showed 22% contact-killing efficacy, which could be

due to the spraying nature of the method, including a drying step that may affect bacterial viability.

The modified JIS method on the other hand, allowed staphylococci to grow on glass slides in

absence of an adsorbed protein film (see also Table 6.2).

Table 6.2 Comparison of the bacterial (S. epidermidis ATCC 12228) contact-killing efficacy (%) achieved in the various methods compared. For each method, the bacterial challenge applied is given per cm2, from which the maximum log reduction that can be achieved is calculated. Percentage efficacy is defined as the log reduction achieved divided by the maximally possible log reduction. ± Signs indicate the SD over triplicate experiments with separately cultured bacteria.

Method Bacterial spray

ASTM E2149 JIS Z 2801

Modified JIS Petrifilm®

Challenge (CFU cm-2) 2.2 x 106 6.0 x 105 2.2 x 104 3.0 x 104 8.8 x 103

Max log reduction 6.3 6.8 4.3 4.5 3.9

Control glass slide

Log reduction 1.4 ± 0.2 0.1 ± 0.1 0.2 ± 0.3 -3.4 a b

Efficacy (%) 22.0 ± 3.1 2.0 ± 2.0 5.0 ± 6.7 -51.0a b

QUAT-coating

Log reduction 3.4 ± 0.3 1.7 ± 0.3 4.3 ± 0.0 -0.7 ± 1.1 3.2 ± 0.3

Efficacy (%) 53 ± 4.7 25 ± 5.1 100 ± 0.0 -16 ± 25.1 82 ± 8.9 a single-fold data. b Numbers of CFUs were too many to count.

With respect to the QUAT-coating itself, it can be observed from Table 6.2 that the JIS and

Petrifilm® methods both indicate a high percentage contact-killing efficacy of 100% and 82%,

respectively. The bacterial spray method resulted in an efficacy of 53%, but here it should be taken

into account that also on the control glass surface a killing efficacy of 22% was observed inherent

to spraying method. The ASTM method showed only 25% contact-killing efficacy, while the

modified JIS method demonstrated growth of bacteria adhering to the cationic surface. However,

compared to the glass control, growth is considerably less on the QUAT-coating (-16% versus -

51% contact-killing efficacy, respectively). Since the ASTM method allows relatively little time for

contact-killing to occur, the ASTM method was also applied as a function of the time during which

a bacterial suspension and slides were shaken together. In Figure 6.2 it can be seen that killing

efficacies increase up to 60 min.

The modified JIS method was also applied when the QUAT-coating was covered with an adsorbed

protein film (see Table 6.3). The presence of an adsorbed protein film in this method attenuated

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the contact-killing ability further, i.e. it increased staphylococcal growth. These observations do not

necessarily imply that contact-killing in presence of an adsorbed protein film will also be attenuated

in case another method would have been applied, particularly since the modified JIS method always

yields growth rather than killing.

Figure 6.2 The staphylococcal (S. epidermidis ATCC 12228) contact-killing efficacy in ASTM E2149 as a function of time. Error bars indicate the SD over triplicate experiments with separately grown bacteria. Data points without error bars represent single-fold data.

Table 6.3 Comparison of the bacterial (S. epidermidis ATCC 12228) contact-killing efficacy (%) achieved in the modified JIS method in absence or presence of adsorbed serum proteins. For details, see heading of Table 6.2.

No adsorption of serum proteins

Adsorption from 10% serum

Adsorption from 50% serum

Challenge (CFU cm-2) 3.0 x 104 3.0 x 104 3.0 x 104

Max log reduction 4.5 4.5 4.5

Control glass slide

Log reduction -3.4 a -3.4 a -4.7 a

Efficacy (%) -51.0 a -50.0 a -69.0 a

QUAT-coating

Log reduction -0.7 ± 1.1 -1.8 ± 1.2 -1.4 ± 0.5

Efficacy (%) -16.0 ± 25.1 -41.0 ± 26.7 -31.0 ± 10.5 a single-fold data

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6.5 Discussion

Cationically coated surfaces constitute the unique feature of killing bacteria upon contact, which

offers a promising alternative for antibiotic-based approaches, particularly considering the rise in

the number of antibiotic resistant strains and species developing.[24-25] Unfortunately, no

ubiquitously accepted method exists to properly evaluate the contact-killing efficacy of contact-

killing coatings. In addition, results may be obscured by the fact that for many coatings reported

on in the literature, it is not clear whether effects are due to leaching of residual antimicrobial

compounds[41-44] from a contact-killing coating or due to contact-killing itself. Here we have

evaluated the contact-killing efficacy of an established, non-leaking contact-killing alkylated

hyperbranched polyurea-PEI coating on glass using one and the same strain in five different

methods (see Table 6.1). Four out of the five methods evaluated demonstrated clear staphylococcal

contact-killing, which varied from 100% in JIS Z 2801 to being indicative of reduced growth in the

modified JIS method. Percentage staphylococcal contact-killing rank as follow: JIS Z 2801 =

Petrifilm® > Bacterial spraying > ASTM E2149 = modified JIS, of which the latter indicating

growth (The differences are statistically significant with respect to the next in ranking at p <0.05,

Student T-test).

Differences obtained between JIS Z 2801 and Petrifilm® are statistically insignificant and the

methods are in fact highly comparable. In both methods bacteria are contacted with a contact-

killing surface within a very small fluid volume, ensuring contact. However, the Petrifilm® method

may be considered slightly more convenient than JIS Z 2801 because surviving bacteria are grown

into countable colonies during contacting. In JIS Z 2801 bacteria have to be dislodged by sonication

after adhering strongly to the cationic coating through electrostatic attraction,[39] which not only

constitutes an additional step with respect to the Petrifilm® method, but possibly also explaining

the higher contact-killing efficacy of JIS Z 2801 as the forceful sonication required may yield

additional killing. Moreover, microscopic observations have shown that in the order of 104 bacteria

per cm-2 are still adhering to a cationic coating after the washing and sonication, which constitutes

50% of the challenge and therewith a major drawback of this method that can be avoided using

the Petrifilm® method. The small fluid volumes in which bacteria and contact-killing surfaces are

brought together, also constitute a possible danger of these two methods, as extremely small

amounts of antimicrobial leachables may easily cause the build-up of a high concentration of

antimicrobial compounds to interfere with contact-killing. Therefore it is advisable to use JIS Z

2801 and Petrifilm® in combination with an agar zone of inhibition assay to ascertain that there is

no release of antimicrobial compounds with demonstrable biological effects (see Figure 6.1 for the

demonstration of absence of release of antimicrobial compounds for the coating employed here).

The spray method is used in several papers[37,45] and often demonstrated 100% contact-killing, while

it yielded only intermediate contact-killing in the current comparison of methods. Self-admitted by

the designers of the spray method,[37] determination of the challenge number of bacteria that

actually come into contact with a coating is hard to establish and can easily be over- or

underestimated and cause erroneous contact-killing efficacy. Also air-drying, although only 2 min,

may cause bacterial-dehydration and possible leakage of antimicrobial compounds in a slinking

volume, therewith contributing to killing.

Methods to compare bacterial contact-killing on cationic surfaces

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The ASTM E2149 method[34] only yielded a small staphylococcal contact-killing efficacy (38%),

although contact-killing efficacies against Escherichia coli after 30 min between 50% to 100% have

been reported for different cationic coatings.[46-47] Experiments carried out as a function of time in

this study demonstrated that killing efficacies did not further increase after 60 min (Figure 6.2).

The advantage of the method is that samples with a variety of shapes and sizes can be used, but

the method fails to control the challenge number of bacteria that actually come into contact with

a surface. Also bacterial enumeration is indirect, since aliquots are taken from the suspension for

CFU analysis, instead of measuring directly on the coated surface. Therewith it is impossible to

directly distinguish between killed and adhering bacteria that are still alive. These drawbacks have

likely been considered quite severe by ASTM and the method was deleted as an official ASTM

method in 2010.

The modified JIS method[36] was developed in order to provide an opportunity to the bacteria to

grow during adhesion to a cationic surface as in the clinical situation, which makes it similar in

principle to the Petrifilm® method. Yet in the modified JIS method, the advantage of growth was

too much: all cationic coatings, as nearly all antimicrobial measures, encounter bacterial individuals

that manage to survive the antimicrobial measures. In the human body this may not necessarily be

a problem, as treatment is often prolonged and the immune system may aid in clearing an infection.

However, in the modified JIS method, survivors are able to grow to the extent that “negative

killing” is measured. Accordingly, the method is only useful in combination with the growth

observed on a non-contact-killing surface such as glass in the current study. Compared with glass,

67% growth reduction was observed on our cationic coating. Interpreting this reduction as contact-

killing, this may yield the conclusion that the modified JIS performs almost comparable with the

original JIS and Petrifilm® methods. Although advocated as an advantage of the method that it

can be applied in the presence of a pre-adsorbed proteinaceous conditioning film, this advantage

is not exclusive to the modified JIS method and can be equally applied in all other methods. In the

current study the modified JIS method was applied in presence of a conditioning film of adsorbed

serum proteins, which further enhanced staphylococcal growth on the cationic coating, or with

respect to the glass non-contact-killing control, reduced contact-killing (see Table 6.3).

Interestingly, on other coatings in presence of a conditioning film of adsorbed salivary proteins, no

reduction in contact-killing was observed,[30,32,48] possibly indicating that staphylococcal growth was

caused by adsorbed serum proteins.

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6.6 Conclusions

This study reveals that depending on the method used, very different results can be obtained in the

evaluation of bacterial contact-killing. We conclude that the Petrifilm® and JIS methods are

preferable: Petrifilm® is most convenient and possibly more reliable. Like all others, these methods

need a complementary assay to exclude killing due to release of antimicrobial compounds, because

even a small release of an antimicrobial compound will have a large influence on bacterial killing in

the small fluid volumes of the assays. The modified JIS method is acceptable, but does not contain

balanced amount of nutrients compared to the Petrifilm® method and should only be used with

respect to a non-contact killing control. ASTM and bacterial spray methods are not reliable, the

main reason being the lack of control over the applied bacterial challenge.

6.7 Acknowledgements

This study was entirely funded by UMCG, Groningen, The Netherlands. H. J. Busscher is also

director of a consulting company SASA BV. The authors declare no potential conflicts of interest

with respect to authorship and/or publication of this article. Opinions and assertions contained

herein are those of the authors and are not construed as necessarily representing views of the

funding organization or their respective employer(s).

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