Minimizing Microbial Survival of Cleanroom Surfaces

Tuesday, March 24, 2020

Introduction

Microbial monitoring in cleanrooms is generally performed using standard, cultivation dependent approaches based on the usage of contact plates or swabs. It is well-established that environmental monitoring methods are limited in their ability to recover organisms from a surface due to inherent limitations with the methods; the selectivity of the culture media and incubation time; the complexities of microbial surface adhesion; and due to the presence of ‘active but non-culturable’ organisms. This places a greater emphasis upon environmental control. One control aspect is with cleanroom surface design.

One design aspect is with the incorporation of antimicrobial materials into surfaces as a means to reduce microbial numbers (or at least to prevent microorganisms from growing). This addition has become commonplace in many hospitals1 and within food factories;2 however, the adoption has been slower within pharmaceuticals (where the use of antibacterial materials used to coat cleanroom surfaces is sometimes referred to as “biotrunking”). A second design aspect lies with the selection of surface properties of materials, so that surfaces can reduce the possibility of microbial attachment, making disassociated organisms easier to kill by disinfection. Combined, antimicrobial surfaces with specific topography has the potential to reduce microbial survival in cleanrooms.

In this article, different antimicrobial technologies together with physical properties are considered together with a review of available literature to examine the efficacy of such surface materials.

Microorganisms on Cleanroom Surfaces

Microorganisms will be present on cleanroom surfaces at different time points and in different numbers, depending on the assigned class of the cleanroom and the challenge population and types of contamination vectors. Most organisms on surfaces will have either settled out from the air, via microbial carrying particles, or they will originate from items introduced into the cleanroom or via direct touching by personnel. Contamination levels can be controlled through controlling the items introduced into the cleanroom; personnel behaviors and gowning; and through cleaning and disinfection of surfaces, although this is a factor of the frequency of application and the efficacy of the disinfection application itself.

Where microorganisms are present these can prove challenging, in terms of survival and ability to be removed. A challenge arises since many human skin commensurate organisms can adapt to extremely low nutrient content and can persist on dry surfaces for a long time.3 In relation to this, adhering to surfaces provides bacteria with many advantages. Attachment to horizontal surfaces stimulates bacterial growth (particularly in nutrient-poor environments) as organic material suspended in liquid settles, is deposited on surfaces, and this increases the local concentration of nutrients available to the microbe.4 A further challenge relates to the mechanisms of bacterial attachment. There are several means by which microorganisms colonize a surface. The four common steps involved are: transport, initial adhesion, bioattachment and colonization. The transportation of a microbial cell to a surface is either by direct physical contact or as a result of gravity, convection or diffusion.

Attachment, which is a factor of physicochemical interactions between microbial cells (specifically hydrodynamic and electrostatic interactions enabling the adhesive force between bacteria and surfaces to increase rapidly)5 and the surface, creates a key divide between laboratory testing of disinfectant efficacy and how disinfecting any surface achieves an equivalent level of kill in the field. The surface interactions vary between some types of bacteria (where the phenomena of attachment is enhanced for those bacteria possessing fimbriae) and some different surfaces can make microorganisms harder to remove than others.6 Removal from the surface is important in the context of ‘cleaning’ with a detergent, which helps to disassociate microorganisms from a surface so they can be more easily killed by the application of a disinfectant. Furthermore, bacterial cells that colonize on surfaces for several days become harder to kill; while biofilms are not likely to form on most standard cleanroom surfaces, cells adhered to surfaces and not associated with biofilms have resistance profiles that are similar to biofilm cells.7

Additional factors are with how microorganisms interact in terms of the age or surface damage to the material and with the presence of residues. Another consideration is with surface wetness, since the transfer from moist surfaces is easier than for dry surfaces.8

These microbial resistant affects can be minimized through good surface design in cleanrooms, as a general cleanroom design concept, and potentially through the incorporation of antimicrobial compounds in surface materials.

Cleanrooms Surface Finish Expectations

There are many factors which help to keep a cleanroom clean, associated with air filtration, air movement, air changes and pressure differentials.9 These aspects of air control need to be supported by overall design and choice of fabric materials (cleanroom architecture). The expectation is that all construction materials have easily cleanable surfaces and the materials selected should not react with cleaning agents or be degradable. The surfaces that contact components, in-process materials or drug products must be non-reactive and not additive or absorptive. Finishes for walls and ceilings within the premises need to be easily cleanable, monolithic and smooth. Moreover, crevices, joints and corners that potentially could collect dirt should be minimalized and all joints to be filled. With equipment inside the cleanroom, not only should this be ‘cleanable’, and have low particle emissions; the equipment should have low electrostatic properties to avoid particles adhering to the equipment (through electrostatic attraction where particles, including microorganisms, are bound onto the surface of equipment instead of remaining airborne).10

In addition to surface design, cleanrooms are best protected if contamination ingress via personnel is minimized; hence, prior to entry into cleanrooms contamination from personnel footwear can be reduced through the use of polymeric flooring or tacky-mats,11 to support the personnel gowning process.

The design features described are well established. The addition of antimicrobial materials to surfaces is less so widespread within pharmaceuticals and healthcare. Nevertheless, there are potential advantages in considering this type of technology.

Incorporation of Antimicrobial Materials

An antimicrobial surface refers to any material that contains an antimicrobial agent that inhibits the ability of microorganisms to grow. Various antimicrobial technologies are available as a range of additive products that have been formulated around an active ingredient. Silver and copper-zinc are commonly used active ingredients due to their efficacious properties and chemical stability, through processes that can incorporate them into materials such as polymers, fabrics, coatings and paper.12 There are two categories of antimicrobial surfaces: totally incorporated biocides such as impregnated materials such as plastics and metals; and the use of surface coatings in the form of paints, or special applications involving spraying, padding or dipping (where the antimicrobial is fixed on and within a thin polymer film, thus providing a biostatic action). The focus in this article is with incorporated antimicrobial compounds.

Silver-Coated materials

Silver has a long history in healthcare settings due to efficacy against a range of microorganisms and lack of toxicity to non-target cells.13 Silver is a bactericidal at minute concentrations, exhibiting an ‘oligodynamic’ effect through the presence of toxic metal ions. The use of silver metal as a bactericidal agent requires the oxidation to the Ag+ ion, which is a slow release process under normal conditions and leads to low effective silver concentrations. Hence silver salts in the form of silver nitrate have been used for different applications.14 As an example, one study of a silver coated material, within a healthcare setting, showed a mean reduction in bacterial counts of 95.8% was demonstrated on the treated surfaces compared with untreated surfaces.15 Such studies show that silver is a broad-spectrum agent: it is bactericidal to a large number of Gram-positive and Gram-negative microorganisms, many aerobes and anaerobes.

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In terms of adding silver-ions or silver nanoparticles to materials suitable for cleanroom use, there are different technologies which can be used to achieve this. One method is by creating an alloy using Active Screen Plasma (ASP), which was developed by the University of Birmingham (UK). ASP technology creates a composite or hybrid metal screen and the combined sputtering, back-deposition and diffusion allows the introduction of silver into a stainless steel surface, along with nitrogen and carbon.16 The silver acts as the bacteria killing agent and the nitrogen and carbon make the stainless steel much harder and durable. As well as cleanroom surfaces, silver has been used in implements like forceps. In addition to silver, other metals have antibacterial properties can be incorporated into surface materials. These include copper and zinc (and alloys like brass).

Organic and Cationic Biocides

Organic or cationic biocides are incorporated into plastic during the manufacturing stage. For plastics, the biocide is added into the manufacturer’s virgin resin, blended, melted, then molded or extruded into the final article. With plastics the process provides a fixed population of the active ingredient on the material surface. When bacteria reach a treated surface, the cell encounters the hydrocarbon portion of the biocidal adjunct. If this is assimilated into the cell, contact with the positively charged nitrogen atom destabilizes the electrical equilibrium within the bacterial cell so that the cells can no longer function correctly and the cell dies.

Copper-Coated Materials

Metals, such as copper, have been used for their antimicrobial properties for thousands of years. This includes the purposes of water disinfection and food preservation, as was practiced by Phoenicians, Greeks, Romans and Egyptians.17 Copper alloy surfaces have intrinsic properties to destroy a wide range of microorganisms. The mechanisms of microbial kill involve the release of copper ions from surfaces. On contact with a microorganism these ions either exhibit direct action (kill) or they lead to the generation of secondary agents of toxicity, including reactive oxygen species, protein dysfunction or membrane damage, affecting several cell targets and which kill the organism. An abundance of peer-reviewed antimicrobial efficacy studies have been conducted around the world regarding the efficacy of copper to destroy Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, influenza A virus, adenovirus, and fungi;18-19 and under conditions of different temperatures and relative humidity.20 This range of organisms has a clinical-setting focus; however, the indicative types of organisms suggest that effectiveness can be achieved against Gram-negative and Gram-positive vegetative organisms, as well as those bacteria capable of forming endospores.

Resistance

As with any antimicrobial chemical-based treatment, there are indications that organisms can develop resistance to the chemical compounds added to surfaces. Some organisms can exhibit resistance to silver and copper and this resistance can develop within microbial populations. There are various mechanisms by which bacteria can exhibit resistance. These include: extracellular sequestration of copper ions; having an impermeable membrane to metal ions; possessing copper-scavenging proteins; and undertaking the active extrusion of ions from the cell.21 Because of this, antimicrobial surfaces cannot be considered as a panacea for the complete elimination of surface deposited organisms and it must be used in conjunction with other contamination control measures like cleaning and disinfection.

Cost

While the antimicrobial properties are reasonably well-established and backed by literature showing microbial kill, most of the current fabrication methods for producing such patterns are still too complex and involve high costs. This has delayed their implementation on a larger scale. The argument for such surfaces should attempt to balance costs against the cost involved with batch rejection, especially for products processed in ISO class 5 / Grade A areas.

Long-Term Use

There have been few studies about the long-term stability of coated materials in the cleanroom setting. With antimicrobials tied into the polymer matrix, this happens in a complex manner that is not completely understood and some migration could occur within polymer interstices and in the amorphous parts of the matrix. It is also unknown what the effect of wear or abrasion is on the surface or how easy such surfaces are to repair or replace. These factors can be addressed over time with stability studies and, hopefully, some published data.

Physical Factors

As well as the incorporation of chemical compounds, there are some physical features that can be applied to surfaces to discourage microbial attachment, such as electrical charge and surface roughness.

Polymeric Materials

Polymeric flooring is an alternative to the conventional tacky-mat, and this is manufactured from a non-toxic, plasticized material and is designed to retain particulate contamination (viable and non-viable) that comes into contact with its surface. A function of polymeric flooring is to attract particles to its surface and retain them for long periods of time (until such a time when they can be removed, totally, through cleaning and disinfection).22

Ultrahydrophobic Coatings

It follows that the surface energy of surfaces plays an important role in microbial adhesion. Different microorganisms will be able to adhere to either hydrophilic or hydrophobic surfaces, depending on species and other interactions. To avoid this, rendering the surface energy to extremely low to create surfaces that are ultrahydrophobic can help to prevent microbial adhesion by causing the repellence of microbes This physiochemical state can be achieved by dip, flow or spin coating with ultrahydrophobic polymers, such as poly(methylpropenoxyfluoroalkylsiloxane) or poly(perfluoroacrylate).23

Surface Roughness

Product contact surfaces in cleanrooms should be smooth enough to be easily cleanable. The smoother the surface, then the lower the possibility of a microorganism attaching to it. The roughness (or smoothness) of a surface is expressed in μm, as an Ra-value, which is quantified by the deviations in the direction of the normal vector of a real surface from its ideal form. This is an important consideration since a high Ra increases the possibility of microbial attachment. There is also an efficiency consideration in that, generally, the cleaning time required increases with surface roughness. The commonly adopted ‘rule’ is that contact surfaces should have a maximum roughness of Ra = 0.8 μm (often the Ra requirement is <0.4μm).24 The standard for assessing surface roughness is ISO 4287:1997.25 To achieve this quality of surface, this is either natural to the material or material may be rendered more acceptable by electropolishing (as with stainless steel) or alternative surface treatments are put in place.

Summary

There are various elements that need to be considered when formulating a contamination control strategy and the choice of surface is an important consideration. As well as long-established criteria, such as cleanability, there are other factors that should now be considered: the active (bacteria killing) or passive (preventing bacteria attachment) properties of cleanroom surfaces are now providing new solutions to effectively reduce microbial survival in the cleanroom environment. As technology advances, an improved understanding of surface–microbe interactions at the micro‐ and nanoscale will enable scientists to fabricate surfaces that will have a more effective microbial kill and prove robust enough to be used in the busy cleanroom environment.

References

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