Figure 1. Mode of action of leaching versus nonleaching antimicrobial polymers, leaching antimicrobial polymers are characterized by antimicrobials (red dots), which are released by the polymer into the surrounding to mediate the antimicrobial effect by a chemical interaction with the germs (green). Hereby, the additive forms a concentration gradient (pink gradient), which can lead to the development of resistant pathogens in sublethal concentrations of the additive. Depending on the used type of a leaching additive a sensitization reaction can occur. Nonleaching antimicrobial polymers have immobilized antimicrobial agents (blue rods) which generate a positively charged surface, which mediate the antimicrobial effect by a physical effect. For this, the germs need direct contact with the material surface. So far, no adverse events are reported.
Proliferation based assays, such as the Certika assay, [ 38,39 ] are designed to measure the antimicrobial efficacy of both, leaching and nonleaching additives, based on the reproduction and release of daughter cells over a period of 18 h after inoculation. Regardless of the antimicrobial mode of action, the implication for bacterial growth on the surface is determined by the release of vital daughter cells, which are responsible for the infection development. The growth activity of these offspring bacteria is then monitored over time. Antimicrobial activity is determined by the time needed to reach a defined optical density (OD) which depends on the number of released cells (Figure 2 ). In comparison to untreated controls, antimicrobially active materials demonstrate their antimicrobial effect by a delayed (shift to the right) or even inhibited growth. A time difference, the so called “net onset OD” of 6 h, represents a reduction of >99.9% (3 log scales) of bacteria on the sample surface. The test is performed in a 96-well plate allowing for simultaneous measurement of eightfold replicates with improved standardization and reproducibility. However, samples have to be prepared to fit into a 96-well plate. This is a unique feature of this test method in that the test is less dependent on sample geometry than other tests and thus, end products can be used for the determination of the antimicrobial efficacy.
The proof of efficacy of antimicrobial products is usually performed with a variety of in vitro test systems. However, most current test systems have been developed for leaching substances and lack reliability for nonleaching products. The Certika test was specifically designed to test the antimicrobial properties of both, leaching and nonleaching antimicrobial products.
In this in vitro study, we investigated the antimicrobial performance of leaching and nonleaching antimicrobial materials in direct comparison with CVCs as an ideal and highly relevant system. For qualitative and quantitative analysis of the antimicrobials performance, we have used the Certika test method, which was established previously. The presented protocol will provide sound quantitative evaluation of other novel antimicrobial materials on implants and is therefore an important step toward efficient disease and hygiene control in the use of materials for medical applications.
The antibacterial effectiveness of antibacterial catheters was tested with the proliferation assay Certika according to ISO DIN EN 17025 (QualityLabs BT GmbH, Nuremberg, Germany). [ 33 ] For the leaching types catheters with coatings of ionized silver (AgION), rifampicine-miconalzole, silver/sulphadiazine/ chlorhexidine, and silver/carbon/platinum were used. Nonleaching catheters included coatings with polyhexanide or poly-guanidine-derivatives.
Catheter samples were either used without a pretreatment or after a pretreatment. As pretreatment the samples were incubated in a 96-well micro titer plate in 200 µL 10% human plasma in phosphate buffered saline (PBS) at 37 °C for 1 h to allow the formation of a plasma conditioning film on the material surface and to thus simulate the in-use application of CVCs. The samples were then washed in 1× PBS pH 7.2 for 10 min. For testing 200 µL bacteria test strains (1 × 106 Colony forming units (CFU) mL −1) were added to each sample and incubated at 37 °C for 1 h to allow bacterial cells to adhere to the sample surface. Loosely bound bacteria were then removed by washing in PBS pH 7.2
Figure 2. Certika data evaluation, A) bacterial growth is optically determined every 30 min over a period of 48 h. The red line shows bacterial growth curve of a typical reference. Bacterial growth of an antimicrobial sample (blue line) appears delayed due to the lower number of vital offspring. Samples, which eliminate all bacteria on the surface, will show no growth curve (green line). The antimicrobial activity is calculated from the time needed to reach an optical density (OD) of 0.2 with an antimicrobially treated sample ( tB), relative to an untreated sample ( tA). A time difference (net onset OD) of 6 h represents a reduction of ≥99.9% (3 log) of bacteria on the material surface, a net onsetof 8 h a reduction of 99.99% (4 log). B) Certika test results of all tested catheters, exemplary shown for one test against Staphylococcus aureus MRSA. All samples are compared to a nonantimicrobial reference catheter (1). The CVC with ionized silver (2) showed only minor efficacy against the tested strain. All other catheters, treated with rifampicin-miconazole (3), silver/sulphadizine/chlorhexidine (4), silver/carbon/platinum (5), polyhexanide (6), and poly-guanidine derivates (7), demonstrated bactericidal activity.
for 10 min. Afterward the samples were incubated in 200 µL of minimum medium (PBS with 1% tryptic soy broth (TSB)) at 37 °C for 18 h (challenge time). After removal of the test samples, each well was supplemented with 50 µL TSB complete medium. The bacterial growth (of the daughter cells) at 37 °C was recorded every 30 min (readout time, Software KC4 3.4, BioTek) for a period of 48 h by OD measurements in a micro titer plate reader at a wavelength of 578 nm. The recording period was determined by the time until the optical content OD 578 reached the value of 0.2. The measurement was conducted as an eightfold replicate measurement. The antimicrobial effectiveness (net onset OD) of an antimicrobial coated sample was calculated as the difference of the onset OD value of the coated samples and the respective nonantimicrobial blank sample: net onset OD value = onset OD value (antimicrobial sample) minus onset OD value (blank sample).In the proliferation assay, the used test bacteria divide once every 30 min which means that after 5 h the net onset OD (in comparison to a blank sample) results in ten duplications/divisions equating to a log reduction of 2 10:1 (= 1024:1) and/or ≈0.1% of the formed daughter cells. Six net onset OD hours equate to 99.9% growth prevention or 3 log scales (compared to the untreated reference sample) and eight net onset OD hours equate to 99.99% (= 4 log scales) growth inhibition, respectively. Thus, the antimicrobial effectiveness is the difference of time which is required for the reference and the antimicrobial coated samples to reach an optical content of 0.2 (Figure 2 ).
Staphylococcus epidermidis DSM 18857, Staphylococcus aureus MRSA EDCC 5247, E. coli DSM 682/ATCC 10536, Enterococcus faecalis DSM 6134, Pseudomonas aeruginosaDSM 939/ATCC 15442, Klebsiella pneumoniae DSM 6135, and Candida albicans DSM 5817/ATCC 10259.
Figure 3. Antimicrobial efficacy of leaching and nonleaching CVC systems against different strains.The CVC samples were tested for antimicrobial efficacy using the Certika test. The CVCs treated with silver/sulphadizine/chlorhexidine (dark gray), silver/carbon/platinum (black),polyhexanide (hatched), and poly-guanidine derivates (lined) mediated antimicrobial performance against all seven tested germs. The CVC with ionized silver (white) failed to reduce 3 log scales of Staphylococcus aureus MRSA, Enterococcus faecalis, and Candida albicans, the CVC with rifampicin-miconazole (light gray) Pseudomonas aeruginosa. A net onset time of 6 h represents a germ reduction of ≥99.9%, a net onset of 8 h a reduction of ≥99.99%. Results of two different charges of each CVC type are shown. Data reported are mean values of eightfold replicate measurements ± SEM.
New materials with bioactive functions become more and more a focus for hygienic and clinical disease control. Due to the variety of available antimicrobial additives, from classical leaching antibiotics to new nonleaching developments, meaningful testing of those properties is a complex task in which many influencing factors have to be considered. Although numerous variations of antimicrobial tests have been developed and used to detect the antibacterial properties of materials, one of the most relevant aspects is to clearly identify suitable test methods which are able to combine test conditions for catheters with different additive antimicrobial properties. Thereby, several points have to be taken into account. The first concerns the ability of an antimicrobial material additive to kill bacteria during the test period. This is an important issue, since kill kinetics differ between the different additives. Thus, the mode of action and the time required to obtain meaningful data have to be considered. Another fundamental aspect is whether the additive is released by the material or the activity is limited to the surface of the material. While releasing systems normally show good activity in all test systems, nonleaching additives can only show their effectiveness in test methods which are able to resolve surface activity. Other test methods will lead to false negative results. A third aspect is the ability of a testing system to test final products and not specially produced test samples. Methods with the ability to test end products should be preferred as parameters may vary between the production of catheters and test samples which may affect the antimicrobial performance.
The established microbiological test methods such as ISO 22196/JIS Z 2801, Adherence, ASTM E 2149, ASTM E 2180, and others are inadequate for testing antimicrobial efficacy of CVCs due to two reasons: (1) the sample
Macromol. Biosci. 2016, DOI: 10.1002/mabi.201500266 © 2016 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.MaterialsViews.com
Figure 4. Antimicrobial efficacy of leaching and nonleaching CVC systems after plasma preincubation. The CVC samples were either used without pretreatment (white bars) or pretreated in 10% human plasma for 1 h at 37 °C (gray bars) prior to testing. Antimicrobial efficacy was tested using the Certika test against the bacteria A) Staphylococcus epidermidis, and B) multiresistant Staphylococcus aureus MRSA. The plasma preincubation had no or only minor inﬂuence on the antimicrobial effectiveness of the CVCs. The CVCs treated with ionized silver, which partly failed to mediate antimicrobial activity without pretreatment also failed after the preincubation in human plasma. A net onset time of 6 h represents a germ reduction of ≥99.9%, a net onset of 8 h a reduction of ≥99.99%. Data reported are mean values of eightfold replicate measurements ± SEM.
geometries requirements for standard test methods cannot be fulfilled with many products and (2) lack of testing of both types of antimicrobial materials, leaching and nonleaching.
In this study, the performance of different antibacterial catheter types was tested in vitro with the proliferation method (Certika test) for their antimicrobial efficacy against typical CRSBI-related gram-positive, gram-negative bacteria, and fungus yeast. With these first examples of benchmark tests using the Certika test method, the equal antimicrobial effect of leaching and nonleaching coated antibacterial catheters could be demonstrated. It was also shown that all catheter components of nonleaching antimicrobial catheters possess antimicrobial activity.
These results have three different implications. First, the Certica method is a suitable instrument to evaluate the antimicrobial properties not only catheter types with leachable antimicrobial additives but also for nonleaching antimicrobial CVCs. Second, these comparative in vitro data demonstrate that surface-active, nonleaching antimicrobial biomaterials produce excellent inhibition of bacterial growth on the catheter surface which is comparable to catheters coated with leachable antimicrobials. Plasma contact had no or only minor impact on the antimicrobial performance for the tested catheter types. Third, the results allow for further clinical evaluation of nonleaching catheters coated with polyhexanide or polyguanidine derivatives to prove their antimicrobial efficacy in in vivo applications in the daily clinical use.
In summary, this was the first comparative in vitro study to demonstrate the antimicrobial performance of CVC types treated with leaching and nonleaching antimicrobial materials (polyhexanide and poly-guanidine) on a quantitative basis. The presented data clearly show the equivalency of nonleaching polymer additives in CVC applications compared to the commonly used leaching antimicrobial technologies. However, by the use of nonleaching antimicrobial systems the undesired side effects associated with the leaching systems can be significantly minimized. The antimicrobial efficacy of nonleaching CVCs was comparable to leachable CVC types against various clinically relevant infection germs, including gram-positive, gram-negative bacteria, and yeast. In addition, the effect was not reduced after contact to human plasma, suggesting an antimicrobial efficacy under in vivo conditions. Further clinical investigations are needed to prove the antimicrobial efficacy of these new nonleaching antimicrobial CVCs in in vivo applications.
In conclusion, these findings represent an initial step to new nonleaching polymers and products, in which general problems with leaching materials, such as release of bioactive substances, loss of antimicrobial activity, and health risks due to biocide leaching, can be minimized. The applicability of these nonleaching antimicrobial materials of other polymer types and materials provides a potent new strategy to fight against device-associated multidrug infections.
Acknowledgements: We thank B.Braun Melsungen AG for providing the experimental poly-guanidine catheter samples for testing.
Received: July 14, 2015; Revised: October 8, 2015; Published online: ; DOI: 10.1002/mabi.201500266
Keywords: antimicrobial polymer material; catheters; in vitro test
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Macromol. Biosci. 2016, DOI: 10.1002/mabi.201500266
The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim