Best of both worlds

Allen Chasteauneuf thinks that to improve infection control a combined hydrophobic and antimicrobial approach is best. How can we achieve this? Look no further than nanotechnology

Hydrophobic surfaces are probably the best examplified by the lotus flower

This article presents a demonstration of the synergistic benefits resulting from the combination of a hydrophobic surface and an actively antimicrobial coating. Such a combination contributes to improved hygiene and infection control strategies and has a consequential impact on the reduction of costs. 

Having a surface that inhibits dirt and organisms adhering such that they can be removed in a benign and environmentally friendly fashion is beneficial.  Surfaces can act as reservoirs for many types of microorganisms1. Whilst not all microorganisms may be of significance to human health, or indeed to food production facilities, preventing their growth and reducing their numbers will significantly reduce the risks associated with any harmful effects.

Hydrophobic surfaces are probably best exemplified by the lotus flower.  Hydrophobic surface coatings mimic the properties of the Lotus plant whose leaves are kept clean by millions of microscopic hairs, which trap air and repel water and dirt2. A droplet of water on an inclined hydrophobic surface does not slide off; it rolls off. When the droplet rolls over a contaminant, the particle is removed from the surface - as a result, the droplet cleans the surface.

Our own evidence supports this. We undertook a repeat soiling and cleaning test on a stainless steel substrate. The samples were soiled repeatedly by applying a solution containing 30g litre-1 bovine serum albumin (BSA) twice and then a mixed artificial organic soilant once. This soiling cycle was repeated three times and then followed by a final application of 30g litre-1 BSA. Between each soiling event, the samples were cleaned for 10 seconds using light manual abrasion under running mains tap water (around 10-15ºC at 0.5 litres minute-1).

On the uncoated surfaces, the appearance deteriorated when subjected to soiling, repeated soiling and washing cycles caused further deterioration of the surface appearance. The stainless steel became increasingly unattractive and the uncoated stainless steel retained significant organic deposits after the soiling / cleaning regime. In contrast, the coated stainless steel samples maintained their appearance and retained very little soiling. They were both visually and microscopically cleaner than the uncoated surfaces. 

Antimicrobial activity was determined after the final soiling / cleaning event using the method described in ISO 22196: 2007 (formerly the Japanese Industrial Standard JIS Z2801: 2000) modified to include a six hour contact interval.

Table 1 shows the results of a comparison between an uncoated stainless steel sample and a stainless steel surface coated with 4MED, an antimicrobial, hydrophobic coating from Nano Hygiene Coatings.

Contact Time

It can be seen from the table that the population of E coli exposed to the 4MED

Sample	0 Hours	6 Hours	24 hours
Stainless steel coated with 4MED	1.5 x 104	1.6 x 104	<1.00
Uncoated stainless steel	1.5 x 104	1.4 x 103	3.0 x 102

coated stainless steel declined by a statistically significant four orders of magnitude over the 24 hours. An effective kill rate of 99.99%. The bacterial population on the uncoated stainless steel declined by one order of magnitude. 

Claims of any antimicrobial coating should be based on realistic exposure scenarios3. That is to say, the tests should be undertaken in the conditions they will be used, or simulating those

Table 1: Activity of uncoated surface and a 4MED coated surface against E Coli (suspended in sterile water). Geometric mean of 3 replicates as colony forming units cm-2

conditions. 4MED was tested simulating the effect of a surface being splashed by several contaminants - a splash of contaminated water, and a splash of contaminated body / animal fluid.

The antimicrobial coating must also be judged on its performance - how long does it take to work? Is it long-lasting?

Contact Time

The standard test used was the ISO 22196:2007 standard mentioned earlier,

Sample	0 Hours	1 Hour	3 Hours	6 Hours	12 Hours	24 Hours
4MED	1.7 x 104	1.2 x 104	4.5 x 101	<1	<1	<1
Control	1.7 x 104	1.7 x 104	1.5 x 104	9.3 x 103	5.8 x 103	1.9 x 103

modified to include the various suspending media and a wider range of contact intervals. For the contaminated water test, a suspension of E coli was prepared using the method described in ISO 22196:2007, but in sterile

Table 2: Activity of 4MED against a ‘splash’ of E Coli (suspended in sterile distilled water). Geometric mean of 3 replicates as colony forming units cm-2

distilled water containing no nutrient broth.  For the contaminated body fluid test an E coli inoculum was prepared using a solution of 1.5g litre-1 vegetable oil and 1.5g litre-1 bovine serum albumin (BSA) solution (oil / protein solution). 

It can be seen from the results in Table 2, that 4MED achieves a fast rate of kill of the bacterial population, resulting in a reduction of ca two point five orders of magnitude in three hours and a reduction to below the limit of detection in six hours.

Contact Time

Regarding the oil/protein test, the results in Table 3 show that the bacterial

Sample	0 Hours	1 Hour	3 Hours	6 Hours	12 Hours	24 Hours
4MED	1.7 x 104	1.2 x 104	1.7 x 104	1.7 x 104	5.4 x 103	2.6 x 102
Control	1.7 x 104	1.9 x 104	1.3 x 104	1.9 x 104	1.8 x 104	5.6 x 104

population declined by around half an order of magnitude within 12 hours and a statistically significant two point five orders of magnitude in 24 hours. The presence of protein-rich soiling agent appears to have slowed down the antimicrobial properties of the coating, but has not

Table 3: Activity of 4MED against  a ‘splash’ of E Coli (suspended in oil / protein solution). Geometric mean of 3 replicates as colony forming units cm-2

inhibited it.

To determine the longevity of 4MED, tests have been undertaken to artificially age the samples, and then subsequently test the antimicrobial efficacy. 

Accordingly, test samples were produced and then processed in a dishwasher at 40ºC with no detergent (13l per cycle). Samples were tested for activity after 60 dishwasher cycles and 120 dishwasher cycles. Antimicrobial activity was determined using ISO 22196 modified to include a wider range of contact intervals. The size of the surviving bacterial population was determined after intervals of 1, 3, 6, 12, and 24 hours. All data was converted to Colony Forming Units cm-2

The populations suspended in sterile distilled water were reduced by four orders

Sample	0 Hour	1 Hour	3 Hours	6 Hours	12 Hours	24 Hours
60 Cycles	1.8 x 104	3.6 x 103	1.2 x 104	4.7 x 100	<1	<1
Control	1.8 104	8.8 x 103	1.3 x 104	1.9 x 104	1.8 x 104	5.6 x 104
120 cycles	1.3 x 104	6.4 x 103	9.9 x 100	1.2 x 100	1.2 x 100	1.5 x 100

of magnitude following six hours contact to below the limit of detection in 24 hours. The fastest kill rate is in the first three hours. The 4MED coated surfaces show a statistically significant difference from the populations exposed to the unfortified coating.

It can be seen from Figure 5 that the kill profile is very similar for before and after artificial ageing,

Table 4. Activity of 4MED washed 60 and 120 times against E Coli  (Suspended in Sterile Distilled Water). Geometric mean of 3 replicates as colony forming units cm-2

demonstrating that the effect is retained even after a relatively severe ageing regime.

The benefits of a hydrophobic coating on a surface have been made clear.   On uncoated surfaces, the appearance reduces as deposits build up and repeated washing and cleaning causes further deterioration of the surface appearance. The coated hydrophobic surface, with no build-up of soil deposits, becomes easy-to-clean. 

The combination of hydrophobicity with the antimicrobial functionality of 4MED produces synergistic benefits. The antimicrobial coating acts quickly with a four order of magnitude reduction in three to six hours, and is also effective against heavy soiling. The antimicrobial coating has been shown to be robust and durable, with effective kill rates under simulated real-life conditions, even  after artificial ageing.

Figure 1: Appearance of a hydrophobic coated stainless steel surface after repeated soiling & cleaning	Figure 2: Appearance of an uncoated stainless steel surface after repeated soiling and cleaning
	Figure 3: Comparison of antimicrobial efficacy before and after artificial ageing by 60 & 120 Dishwasher cycles

Reducing the growth of bacteria and increasing their rate of decline contributes to a lowering of the risks associated with their harmful effects.  The implications for improved cleaning and hygiene strategies are clear -  4MED makes the cleaning job easier and safer. It can have a direct impact on the costs associated with the cleaning task, contributing to the lowering of risks associated with their harmful effects.